Programmable protein circuits in living cells

ABSTRACT

Some embodiments of the systems, methods and compositions provided herein relate to a compound protease. In some embodiments, the compound protease includes a protease domain and a cut site for another enzyme. In some embodiments, the compound protease includes an association domain. In some embodiments, the compound protease is part of a protein circuit.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional ApplicationNo. 62/619,001, filed Jan. 18, 2018; and U.S. Provisional ApplicationNo. 62/688,859, filed Jun. 22, 2018. The entire contents of theseapplications are hereby expressly incorporated by reference in theirentireties.

STATEMENT REGARDING FEDERALLY SPONSORED R&D

This invention was made with government support under Grant No,HR0011-17-2-0008 awarded by DARPA. The government has certain rights inthe invention.

FIELD

Some embodiments of the systems, methods and compositions providedherein relate to a compound protease. In some embodiments, the compoundprotease includes a protease domain and a cut site for another enzyme.In some embodiments, the compound protease includes an associationdomain. In some embodiments, the compound protease is part of a proteincircuit.

BACKGROUND

Synthetic biology may enable design of new functions in living cells.Many natural cellular functions are implemented by protein-levelcircuits, in which proteins specifically modify each other's activity,localization, or stability. Synthetic protein circuits could provideadvantages over gene regulation circuits in enabling the design of newfunctions in living cells.

SUMMARY

Some embodiments relate to a compound protease, the compound proteasecomprising: a) a protease domain comprising: a first part of theprotease domain, and a second part of the protease domain, wherein whenthe first part and the second part of the protease domain are associatedtogether, they form an active protease domain, and wherein the firstpart and the second part of the protease domain do not self-associate ontheir own to form the active protease domain; b) a cut site, wherein thecut site comprises: a first part of the cut site, wherein the first partof the cut site is linked to the first part of the protease domain; anda second part of the cut site, wherein the second part of the cut siteis linked to the second part of the protease domain, wherein when thefirst and second parts of the cut site are associated together they forman active cut site for an enzyme, and wherein when the active cut siteis cut by the enzyme, the first and second parts of the cut sitedissociate from one another; and c) an association domain, theassociation domain comprising: a first part of the association domainthat is conjugated to the second part of the cut site; a second part ofthe association domain that is linked to the second part of the proteasedomain, wherein the association domain is configured to stabilize theactive protease domain. In some embodiments, the first and second partsof the association domain of the compound protease comprise separatepeptide strands that hybridize together. In some embodiments, the firstand second parts of the association domain of the compound protease area single peptide strand.

Some embodiments relate to a compound protease, the compound proteasecomprising: a) a protease domain comprising: a first part of theprotease domain, and a second part of the protease domain, wherein whenthe first part and the second part of the protease domain are associatedtogether, they form an active protease domain, and wherein the firstpart and the second part of the protease domain do not self-associate ontheir own to form the active protease domain; b) a cut site, wherein thecut site comprises: a first part of the cut site, wherein the first partof the cut site is linked to the first part of the protease domain; anda second part of the cut site, wherein the second part of the cut siteis linked to the second part of the protease domain, wherein when thefirst and second parts of the cut site are associated together they forman active cut site for an enzyme, and wherein when the active cut siteis cut by the enzyme, the first and second parts of the cut sitedissociate from one another; c) a first peptide connecting the firstpart of the protease domain to the first part of the cut site; and d) asecond peptide connecting the second part of the protease domain to thesecond part of the cut site, wherein the first and second linkers areconfigured to stabilize the active protease domain. In some embodiments,the first peptide connecting the first part of the protease domain tothe first part of the cut site comprises a linker. In some embodiments,the second peptide connecting the second part of the protease domain tothe second part of the cut site comprises a linker.

Some embodiments relate to a method, comprising: providing a reactionsolution with the compound protease and the enzyme; and subjecting thereaction solution to a condition that allows the enzyme to cleave thecut site of the compound protease.

Some embodiments relate to a synthetic protein circuit, comprising: afirst protease; and a second protease comprising a cut site specific forthe first protease, wherein the second protease is inactivated bycleavage of the cut site specific for the first protease. Someembodiments comprise a target protein comprising: a degron of the targetprotein that destabilizes the target protein when present on the targetprotein by enhancing degradation of the target protein, and a cut sitespecific for the second protease, wherein the target protein isconfigured to be stabilized or destabilized by cleavage of the cut sitespecific for the second protease.

In some embodiments of the synthetic protein circuit, the first proteaseand the second protease each comprise an HCV protease, a TEV protease,or a TVMV protease.

In some embodiments of the synthetic protein circuit, the secondprotease comprises a first cleavage domain and a second part of thecleavage domain, the first part connecting to the cut site specific forthe first protease, and the second part connecting to another cut sitespecific for the first protease, the second protease's two cut sitesspecific for the first protease each connecting to an association domainof the second protease such as a leucine zipper. In some embodiments,the second protease's two cut sites specific for the first protease eachconnect to a separate association domain of the second protease, whereinthe second protease is active when the separate association domains bindtogether, and wherein the second protease is configured to bedeactivated by cleavage of either of its two cut sites specific for thefirst protease. In some embodiments, one of the second protease'sassociation domains comprises a complementary association domain such asleucine zipper that is complementary to the other association domain ofthe second protease. In some embodiments, the second protease's two cutsites specific for the first protease each connect to a singleassociation domain of the second protease, and wherein the secondprotease is configured to be deactivated by cleavage of either of itstwo cut sites specific for the first protease.

In some embodiments of the synthetic protein circuit, the first proteasecomprises an association domain of the first protease that binds to acomplementary association domain of the second protease, therebyenhancing the first protease's ability to cleave a cut site specific tothe first protease on the second protease.

Some embodiments of the synthetic protein circuit comprise a third,fourth, fifth, sixth, seventh, eighth, ninth and/or tenth protease, eachprotease comprising a cut site specific to at least one of theproteases, and wherein each protease is configured to be destabilized ordeactivated by cleavage of its cut site.

In some embodiments of the synthetic protein circuit, the targetprotein's cut site specific to the second protease comprises a firstpart of the cut site of the target protein and a second part of the cutsite of the target protein, the first part of the cut site of the targetprotein connecting to a domain or motif of the target protein, and thesecond part of the cut site of the target protein connecting to thedegron of the target protein, and wherein the target protein isstabilized by cleavage of its cut site specific for the second protease.

In some embodiments of the synthetic protein circuit, the degron of thetarget protein comprises a masking peptide that connects to the degronof the target protein and blocks cleavage of the target protein's cutsite specific for the second protease, wherein the masking peptide ofthe degron of the target protein comprises the target protein's cut sitespecific for the second protease, and wherein the target protein isconfigured to be destabilized by cleavage of its cut site specific forthe second protease, wherein cleavage of the target protein's cut sitespecific for the second protease uncovers the target protein's degron.

In some embodiments of the synthetic protein circuit, the target proteincomprises a protease, a reporter protein, a fluorescent protein, ascaffold, an actuator protein, a transcriptional regulator, or asignaling protein.

Some embodiments relate to a synthetic protein circuit, comprising: afirst protease, optionally comprising an association domain of the firstprotease; a second protease, optionally comprising a complementaryassociation domain of the second protease; and a target proteincomprising a degron of the target protein that destabilizes the targetprotein when present on the target protein by enhancing degradation ofthe target protein; wherein the target protein is configured to interactwith the first protease, the second protease, a third protease and/or afourth protease to form an OR, AND, NOR, NAND, IMPLY, NIMPLY, XOR orXNOR logic gate.

In some embodiments of the synthetic protein circuit, the target proteincomprises a cut site specific for the first protease and a cut sitespecific for the second protease between the degron of the targetprotein and a part of the target protein, and wherein the target proteinis stabilized by cleavage of either of its cut sites.

In some embodiments of the synthetic protein circuit, the target proteincomprises a cut site of the target protein specific for the firstprotease between the degron of the target protein and a part of thetarget protein, and a cut site specific for the second proteaseconnected to another degron of the target protein and an optionalassociation domain of the target protein, and wherein the target proteinis stabilized by cleavage of both of its cut sites.

Some embodiments of the synthetic protein circuit comprise: a thirdprotease comprising: a cut site specific for the first protease, a cutsite specific for the second protease, and an optional associationdomain of the third protease, wherein the third protease is configuredto be deactivated by cleavage of either of its cut sites; and whereinthe target protein comprises a cut site specific for the third proteasebetween the degron of the target protein and a part of the targetprotein, wherein the target protein is stabilized by cleavage of its cutsite specific for the third protease. In some embodiments, the thirdprotease comprises a first domain of the third protease and a seconddomain of the third protease; wherein the first domain of the thirdprotease comprises the third protease's cut sites specific for the firstand second proteases and the optional association domain of the thirdprotease; wherein the second domain the third protease comprises anothercut site specific for the first protease, another cut site specific forthe second protease, and an optional complementary association domainthe third protease; and wherein the third protease is configured to bedeactivated by cleavage of any of its cut sites.

Some embodiments of the synthetic protein circuit comprise: a thirdprotease comprising a cut site specific for the first protease, andconfigured to be deactivated by cleavage of its cut site; and a fourthprotease comprising a cut site specific for the second protease, andconfigured to be deactivated by cleavage of its cut site; wherein thetarget protein comprises a cut site specific for the third and fourthproteases between the degron of the target protein and a part of thetarget protein, wherein the target protein is stabilized by cleavage ofits cut site. In some embodiments, the third protease comprises a firstdomain of the third protease, a second domain of the third protease, andan optional complementary association domain of the third protease;wherein the first domain of the third protease comprises the cut sitespecific for the first protease; wherein the second domain of the thirdprotease comprises another cut site specific for the first protease;wherein the complementary association domain the third proteaseoptionally comprises two parts of the third protease, each part, thethird protease connected to one of the third protease's cut sites; andwherein the third protease is configured to be deactivated by cleavageof either of its cut sites.

In some embodiments of the synthetic protein circuit, the fourthprotease comprises a first domain of the fourth protease, a seconddomain of the fourth protease, and an optional association domain of thefourth protease; wherein the first domain of the fourth proteasecomprises the cut site specific for the second protease; wherein thesecond domain of the fourth protease comprises another cut site specificfor the second protease; wherein the association domain of the fourthprotease optionally comprises two parts, each part connected to one ofthe fourth protease's cut sites; and wherein the fourth protease isconfigured to be deactivated by cleavage of either of its cut sites.

Some embodiments of the synthetic protein circuit comprise: a thirdprotease comprising a cut site specific for the second protease, andconfigured to be deactivated by cleavage of its cut site; wherein thetarget protein comprises a cut site specific for the first protease anda cut site specific for the third protease between the degron of thetarget protein and a part of the target protein, and wherein the targetprotein is stabilized by cleavage of either cut sites. In someembodiments, wherein the third protease comprises a first domain, asecond domain, and an optional association domain; wherein the firstdomain of the third protease comprises the third protease's cut sitespecific for the second protease; wherein the second domain of the thirdprotease comprises another cut site specific for the second protease;wherein the association domain of the third protease optionallycomprises two parts of the third protease, each part of the thirdprotease connected to one of the third protease's cut sites; and whereinthe third protease is configured to be deactivated by cleavage of eitherof its cut sites.

Some embodiments of the synthetic protein circuit comprise: a thirdprotease comprising a cut site specific for the first protease, andconfigured to be deactivated by cleavage of its cut site; wherein thetarget protein comprises a cut site specific for the third proteasebetween the degron and a part of the target protein, and a cut sitespecific for the second protease connected to another degron of thetarget protein and an optional association domain of the target protein,and wherein the target protein is stabilized by cleavage of both of itscut sites. In some embodiments, the third protease comprises a firstdomain of the third protease, a second domain of the third protease, andan optional complementary association domain of the third protease;wherein the first domain of the third protease comprises the cut sitespecific for the first protease; wherein the second domain of the thirdprotease comprises another cut site specific for the first protease;wherein the complementary association domain of the third proteaseoptionally comprises two parts of the third protease, each part of thethird protease connected to one of the third protease's cut sites; andwherein the third protease is configured to be deactivated by cleavageof either of its cut sites.

Some embodiments of the synthetic protein circuit comprise: a secondtarget protein comprising a degron of the second target protein thatdestabilizes the second target protein when present on the second targetprotein; wherein the target protein comprises a cut site specific forthe first protease between its degron and a part of the target protein,an other degron of the target protein, and a cut site specific for thesecond protease connected to the other degron of the target protein,wherein the target protein is destabilized by its first degron unlessits cut site specific for the first protease is cleaved by the firstprotease, and wherein the target protein is destabilized by cleavage ofits cut site specific for the second protease; and wherein the secondtarget protein comprises a cut site specific for the second proteasebetween its degron and the part of the second target protein, an otherdegron of the second target protein, and a cut site specific for thefirst protease connected to the other degron of the second targetprotein, wherein the second target protein is destabilized by its firstdegron unless its cut site specific for the second protease is cleavedby the second protease, and wherein the second target protein isdestabilized by cleavage of its cut site specific for the firstprotease. In some embodiments, the second target protein comprises acomplementary association domain of the second target protein connectedat or near the other degron of the second target protein or the secondtarget protein's cut site specific for the first protease. In someembodiments, the target protein's other degron comprises a maskingpeptide of the other degron of the target protein connected to thetarget protein's other degron, wherein the masking peptide of the otherdegron of the target protein prevents the target protein's other degronfrom destabilizing the target protein when the masking peptide of theother degron of the target protein is present on the target protein,wherein the masking peptide of the other degron of the target protein isconfigured to be cleaved from the target protein when the targetprotein's cut site specific for the second protease is cleaved by thesecond protease, wherein the target protein is configured to bedestabilized by cleavage of its cut site specific for the secondprotease, wherein cleavage of the target protein's cut site specific forthe second protease uncovers the target protein's other degron therebydestabilizing the target protein. In some embodiments, the second targetprotein's other degron comprises a masking peptide of the other degronof the second target protein connected to the second target protein'sother degron, wherein the masking peptide of the other degron of thesecond target protein prevents the second target protein's other degronfrom destabilizing the second target protein when the masking peptide ofthe other degron of the second target protein is present on the secondtarget protein, wherein the masking peptide of the other degron of thesecond target protein is configured to be cleaved from the second targetprotein when the second target protein's cut site specific for the firstprotease is cleaved by the first protease, wherein the second targetprotein is configured to be destabilized by cleavage of its cut sitespecific for the first protease, wherein cleavage of the second targetprotein's cut site specific for the first protease uncovers the secondtarget protein's other degron thereby destabilizing the second targetprotein.

Some embodiments of the synthetic protein circuit comprise: a thirdprotease comprising a cut site specific for the first protease, a cutsite specific for the second protease, and one or more optionalassociation domains of the third protease, wherein the third protease isconfigured to be deactivated by cleavage of either of its cut sites;wherein the target protein comprises a second degron of the targetprotein, a cut site specific for the first protease, a cut site specificfor the second protease, and two cut sites specific for the thirdprotease, and wherein the target protein is stabilized by cleavage of:its cut site specific for the first protease and its cut site specificfor the second protease, or both of its cut sites specific for the thirdprotease.

In some embodiments of the synthetic protein circuit, the third proteasecomprises a first domain of the third protease and a second domain ofthe third protease; wherein the first domain of the third proteasecomprises the cut sites specific for the first and second proteases andthe optional association domain of the third protease; wherein thesecond domain of the third protease comprises another cut site specificfor the first protease, another cut site specific for the secondprotease, and an optional complementary association domain of the thirdprotease; and wherein the third protease is configured to be deactivatedby cleavage of any of its cut sites. In some embodiments, the targetprotein's cut site specific for the first protease and one of the targetprotein's two cut sites specific for the third protease separate thetarget protein's first degron from a part of the target protein; andwherein the target protein's cut site specific for the second proteasethe other of the two cut sites specific for the third protease, and theassociation domain of the target protein separate the target protein'ssecond degron from the part of the target protein.

Some embodiments relate to a system such as a synthetic protein circuit,comprising: a first protease; a second protease; and target proteinseach comprising: a first degron of the target protein that destabilizesthe target protein when present on the target protein by enhancingdegradation of the target protein, a cut site specific for the firstprotease between the degron of the target protein and a part of thetarget protein, wherein the target protein is configured to bestabilized by cleavage of its cut site specific for the first protease,and a cut site specific for the second protease connected to anotherdegron of the target protein, wherein the target protein is configuredto be destabilized by cleavage of the cut site specific for the secondprotease regardless of whether the first degron of the target protein ispresent on the target protein. In some embodiments, the other degron ofeach target protein comprises a conditional N-end degron such as anN-end degron that is conditional on cleavage of the cut site specificfor the second protease. Some embodiments comprise a third proteasecomprising a cut site specific for the second protease, wherein thethird protease is configured to be deactivated by cleavage of its cutsite specific for the second protease; and wherein the second proteasecomprises a cut site specific for the third protease, wherein the secondprotease is configured to be deactivated by cleavage of its cut sitespecific for the third protease. In some embodiments, the secondprotease comprises a first domain of the second protease, a seconddomain of the second protease, a first complementary association domain,and an optional second complementary association domain of the secondprotease connected to the first or second domain of the second protease;wherein the first domain of the second protease comprises the cut sitespecific for the third protease; wherein the second domain of the secondprotease comprises another cut site specific for the third protease;wherein the first complementary association domain of the secondprotease optionally comprises two parts of the complementary associationdomain of the second protease, each part of the complementaryassociation domain of the second protease connecting to one of thesecond protease's cut sites specific for the third protease; and whereinthe second protease is configured to be deactivated by cleavage ofeither of its cut sites. In some embodiments, the third proteasecomprises an optional association domain of the third protease, andwherein cleavage of the third protease's cut site by the second proteaseremoves at least part of a cleavage domain of the third protease,thereby deactivating the third protease. In some embodiments, thestability of the target proteins comprises an analog behavior that isdependent on a concentration of the first protease, wherein a higherconcentration of the first protease has a greater stabilizing effect onthe target proteins than a lower concentration of the first protease. Insome embodiments, the stability of the target proteins comprises ananalog behavior that is dependent on a concentration of the secondprotease, wherein a higher concentration of the second protease has agreater destabilizing effect on the target proteins than a lowerconcentration of the second protease. In some embodiments, theconcentration of the second protease is decreased by a higherconcentration of the third protease as compared to a lower concentrationof the third protease or by a higher amount of a nucleic acid encodingthe third protease as compared to a lower amount of a nucleic acidencoding the third protease. In some embodiments, the analog behavior ofthe target protein that is dependent on a concentration of the secondprotease is more sharp and/or comprises a greater threshold fordestability of the target protein at a higher concentration of the thirdprotease as compared to a lower concentration of the third protease, orat a higher amount of a nucleic acid encoding the third protease ascompared to a lower amount of a nucleic acid encoding the thirdprotease. In some embodiments, the analog behavior of the target proteincomprises a bandpass behavior. In some embodiments, the first proteasecomprises a first domain of the first protease and a second domain ofthe first protease; wherein the first domain of the first proteaseconnects to a first conditional dimerization domain of the firstprotease; wherein the second domain of the first protease connects to asecond conditional dimerization domain of the first protease; whereinthe first and second conditional dimerization domains of the firstprotease are configured to dimerize with each other upon binding adimerizing agent. In some embodiments, the conditional dimerizationdomains of the first protease each comprise one of an FK506 bindingprotein (FKBP), GyrB, GAI, Snap-tag, eDHFR, BCL-xL, CalcineurinA (CNA),CyP-Fas, FRB domain of mTOR, GID1, HaloTag, and/or Fab (AZ1). In someembodiments, the dimerizing agent comprises FK1012, FK506, FKCsA,Rapamycin, Coumermycin, Gibberellin, HaXS, TMP-HTag, or ABT-737.

Some embodiments relate to a method of activating a signaling pathway ina cell, comprising providing to the cell a synthetic protein circuit ora nucleic acid encoding the synthetic protein circuit, the syntheticprotein circuit comprising: a protease comprising a first part of theprotease and a second part of the protease, the first part of theprotease connecting to a signaling protein, and the second part of theprotease connecting to a binding protein that binds to an activated formof the signaling protein, wherein the first part and the second part areconfigured to form an active protease when the binding protein binds tothe activated form of the signaling protein; and an effector proteincomprising a cut site specific for the protease, wherein the effectorprotein configured to be activated by cleavage of its cut site specificfor the protease.

In some embodiments of the method, the synthetic protein circuitcomprises a second protease that inactivates the first protease and/orthe effector protein. In some embodiments, the signaling pathwaycomprises a cell death pathway. In some embodiments, the signalingprotein comprises a signal transduction protein such as Ras or afragment thereof. In some embodiments, the binding protein comprises Rafor a fragment thereof such as a Ras-binding domain (RBD). In someembodiments, the effector protein comprises a protease or cell deathprotein such as a caspase.

Some embodiments relate to a nucleic acid encoding all or a portion of asynthetic protein circuit as described herein. In some embodiments, thenucleic acid comprises DNA. In some embodiments, the DNA comprises avector configured for transient expression in a cell. In someembodiments, the DNA comprises an expression construct configured tointegrate into a host cell's DNA. In some embodiments, the nucleic acidcomprises RNA such as an mRNA.

Some embodiments relate to a compound protease, the compound proteasecomprising: a) a protease domain comprising: a first part of theprotease domain, and a second part of the protease domain, wherein whenthe first part and the second part of the protease domain are associatedtogether, they form an active protease, and wherein the first part andthe second part of the protease domain do not self-associate on theirown to form the active protease; and b) a cut site, wherein the cut sitecomprises: a first part of the cut site, wherein the first part of thecut site is linked to the first part of the protease domain; and asecond part of the cut site, wherein the second part of the cut site islinked or indirectly connected to the second part of the proteasedomain, wherein when the first and second parts of the cut site areassociated together they form an active cut site for an enzyme, andwherein when the active cut site is cut by the enzyme, the first andsecond parts of the cut site dissociate from one another.

In some embodiments of the compound protease, the first part of the cutsite is covalently linked to the first part of the protease domain by afirst peptide linkage, and/or wherein the second part of the cut site iscovalently linked to the second part of the protease domain by a secondpeptide linkage. In some embodiments, the first peptide linkagecomprises a linker peptide comprising 1-10, 10-25, 25-50, 50-100, or100-1000 amino acids. In some embodiments, the second peptide linkagecomprises a linker peptide comprising 1-10, 10-25, 25-50, 50-100, or100-1000 amino acids.

In some embodiments of the compound protease, the second part of theprotease domain comprises a part of an association domain connected tothe second part of the protease domain, wherein the part of theassociation domain connected to the second part of the protease domainis configured to recruit the enzyme to the active cut site by binding asecond part of the association domain on the enzyme.

Some embodiments of the compound comprise a second cut site, wherein thesecond cut site comprises: a first part of the second cut site, whereinthe first part of the second cut site is linked to the second part ofthe protease domain; and a second part of the second cut site, whereinthe second part of the second cut site is linked or indirectly connectedto the first part of the protease domain; wherein when the first andsecond parts of the second cut site are associated together they form anactive second cut site for the enzyme, and wherein when the activesecond cut site is cut by the enzyme, the first and second parts of thesecond cut site dissociate from one another. Some embodiments comprisean association domain the association domain comprising: a first part ofthe association domain, conjugated to the second part of the first cutsite; a second part of the association domain, conjugated to the secondpart of the second cut site, wherein the association domain is configureto stabilize the active protease domain. In some embodiments, the firstpart of the cut site is covalently linked to the first part of theprotease domain by a first peptide linkage, and/or wherein the firstpart of the second cut site is covalently linked to the second part ofthe protease domain by a second peptide linkage. In some embodiments,the first peptide linkage comprises a linker peptide comprising 1-10,10-25, 25-50, 50-100, or 100-1000 amino acids. In some embodiments, thesecond peptide linkage comprises a linker peptide comprising 1-10,10-25, 25-50, 50-100, or 100-1000 amino acids. In some embodiments, thesecond part of the cut site is indirectly connected to the second partof the protease domain through the association domain, wherein the firstand second parts of the association domain are covalently ornon-covalently linked together.

Some embodiments of the compound protease comprise an association domainof the compound protease comprising a first part and a second part,wherein the first part of the association domain links to the secondpart of the first cut site, and wherein the second part of theassociation domain links to the second part of the second cut site. Insome embodiments, the first part of the cut site is covalently linked tothe first part of the protease domain by a first peptide linkage, and/orwherein the first part of the second cut site is covalently linked tothe second part of the protease domain by a second peptide linkage. Insome embodiments, the first peptide linkage comprises a linker peptidecomprising 1-10, 10-25, 25-50, 50-100, or 100-1000 amino acids. In someembodiments, the second peptide linkage comprises a linker peptidecomprising 1-10, 10-25, 25-50, 50-100, or 100-1000 amino acids. In someembodiments, the second part of the cut site is indirectly connected tothe second part of the protease domain through the association domain(for example, as in FIGS. 1A and 1B). In some embodiments, theassociation domain connecting to the second part of the cut site and tothe to the second part of the second cut site is configured to recruitthe enzyme to the active cut site and/or to the active second cut siteby binding a second part of the association domain on the enzyme.

Some embodiments relate to system such as a synthetic protein circuit,comprising: a first protease; a second protease; and a target proteincomprising: one or more cut sites specific for a first, second, and/orthird protease, and a degron of the target protein configured tostabilize or destabilize the target protein based on its configurationwith one or more of the target protein's cut sites specific for thefirst, second, and/or third proteases. In some embodiments, the firstprotease comprises a first domain of the first protease and a seconddomain of the first protease; wherein the first domain of the firstprotease connects to a first conditional dimerization domain of thefirst protease; wherein the second domain of the first protease connectsto a second conditional dimerization domain of the first protease;wherein the first and second conditional dimerization domains of thefirst protease are configured to dimerize with each other upon binding adimerizing agent.

In some embodiments of the synthetic protein circuit or method, the/afirst protease, second protease, third protease, and/or fourth proteasecomprises a compound protease as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C depict nonlimiting examples of compound proteases asdescribed herein.

FIGS. 1D-1J depict information relating to design of composable proteincircuit components of some embodiments. FIG. 1D, Composable proteinunits can regulate one another in arbitrary configurations with diversefunctions (middle). Protein-level circuits can interface directly withendogenous protein pathways and operate without modifying the genome orentering the nucleus. (right). FIG. 1E, The protease-activatablereporter can be stabilized by removal of a DHFR degron through proteasecleavage of a corresponding target site. TMP inhibits the degron, andthus stabilizes the reporter. Middle, flow cytometry distributions ofreporter fluorescence with or without TEVP. Distributions are limited tothe gated area in FIG. 5A. Solid curves indicate skew Gaussian fits.Vertical dashed lines and stars indicate distribution modes, which areplotted in subsequent figure panels. Right, analysis of reporterresponse to TMP and/or TEVP. Each dot represents one replicate. Starsindicate data from middle panel. FIG. 1F, In the protease-repressiblereporter, protease cleavage exposes an N-end degron (covered target) todestabilize reporter. FIG. 1G, Three proteases (columns) exhibitorthogonal regulation of three reporters (rows). Mean fluorescentintensity of 3 independent measurements were normalized to theTMP-stabilized value of its corresponding reporter. FIG. 1H, Design forsome protease-repressible proteases. TEVP was split as indicated andthen reconstituted through dimerizing leucine zippers. Aleucine-zipper-tagged HCVP can dock with the target TEVP and cleave itto remove leucine zippers, effectively repressing TEVP. TVMVP can beregulated using the same design. FIG. 1I, A single-chain variant of theHCV-repressible TEVP allows docking of, and repressive cleavage by,HCVP. FIG. 1J, Protease regulation can propagate through a three-stagecascade. Repressible HCVP uses a variant design, in which TEVP cleavageseparates core HCVP from its docking leucine zipper andactivity-enhancing co-peptide.

FIG. 1K depicts a legend for the symbols shown in FIGS. 1A-1J and inother Figures.

FIG. 1L depicts some examples of protein circuits and their use.

FIGS. 2A-2I depict example CHOMP circuits implementing binary logicgates in accordance with some embodiments. For each indicated gate, TEVPand HCVP served as binary inputs, which were either included or excludedin transfections. Citrine fluorescence serves as gate output. The designand performance of each non-trivial two-input logic gate is shown fortriplicate experiments (black dots). Fluorescent intensity in each panelwas normalized to the corresponding reporter stabilized with TMP (forgates containing only C-terminal degrons) or Shield-1+TMP (for gatescontaining degrons at both termini). Grey regions indicate range frommaximum “OFF” value to minimum “ON” value for that gate.

FIGS. 3A-3H depict information relating to bandpass filtering and pulsegeneration circuits according to some embodiments. FIG. 3A, For bandpassfiltering, the expression of co-regulated inputs TEVP and TVMVP werecontrolled by the amount of transfected DNA, or by doxycycline (square)induction. The amount of HCVP plasmid can be varied to tune therepression arm. FIG. 3B, Input-output curve of the activation arm in theabsence of TVMVP. Here and in subsequent panels, dots indicate duplicatemeasurements, and curve is a model fit. FIG. 3C, Input-output curve ofthe repression arm, in the presence of constant TEVP and increasinglevels of HCVP, which increased the repression threshold and sharpensthe response. FIG. 3D, Bandpass behavior of a complete circuit.Increasing HCVP expression shifted the position and increases theamplitude of the peak response. Data in FIGS. 3B-3D were normalized tothe TMP-stabilized reporter. FIG. 3E, Delayed repression can enablepulse generation. In this design, rapamycin-induced dimerization of FKBPand FRB domains reconstituted TEVP. Cleavage of the reporter by TEVPallowed maturation of far-red fluorescent protein (IFP, FIG. 8D). FIG.3F, The pulse circuit was completely encoded on a single transcript,with protein components (indicated) separated by “self-cleaving”sequences (T2A, P2A). FIG. 3G, Filmstrips of a single cell stablyincorporating both the pulse generation circuit, as well as aconstitutive Cerulean segmentation marker. After rapamycin induction(t=0), the output IFP signal increased and then decayed, while Ceruleansignal remained constant. FIG. 3H, Traces of IFP fluorescence in 24individual cells. This analysis omitted cells that exhibitedphototoxicity or moved out of the field of view. Black line indicatesmedian fluorescence over all cells at each time point.

FIG. 3I depicts a non-limiting example of a synthetic protein circuit inaccordance with some embodiments.

FIGS. 4A to 4E depict information relating to example CHOMP circuitsthat enable conditional activation of Casp3 in Ras-activating cells.FIG. 4A, The core circuit (left) links Ras activation by SOS_(CA) orEGFRvIII to Casp3 activation. The full circuit (right) incorporates anadditional TVMVP component to enhance selectivity. New regulatoryfeatures introduced in this circuit are explained schematically incorresponding numbered boxes. Box 1, input from upstream activators ofRas such as SOS_(CA) and EGFRvIII activates Ras, causing it to bind RBD,reconstituting RasTEVP. Box 2, Engineered Casp3 tagged with a membranelocalization sequence (“mts”) can be converted from an inactive to anactive state by TEVP cleavage. Box 3, TVMVP cleavage detaches Casp3 fromthe membrane, reducing its ability to be activated by membrane-localizedTEVP. FIG. 4B, TEVP activates the engineered Casp3, while TVMVP inhibitsthis activation. Cells transfected with indicated components wereanalyzed to determine the reduction index (percentage of cell numberreduction compared to cells transfected with only a fluorescent marker,see FIG. 9B). FIG. 4C, The core circuit preferentially reduced cellnumber in the presence of ectopic SOS_(CA). The full circuit exhibitedimproved selectivity. FIG. 4D, The full circuit (top diagram) and apositive control circuit incorporating a G12V mutation that makes Rasconstitutively active and a C152A mutation that abolishes TVMVP activity(bottom diagram) were each encoded as a single transcript. FIG. 4E, In amixed population, the single-transcript circuit (FIG. 4D, top)conditionally reduced the number of EGFRvIII cells (left) and SOS_(CA)cells (right) compared to that of co-cultured control cells. Thepositive control circuit (FIG. 4D, bottom) reduced the number of bothfractions. Dashed line indicates the upper limit of reduction indexmeasured with the positive control circuit.

FIG. 4F-4I depict non-limiting examples of synthetic protein circuits inaccordance with some embodiments.

FIGS. 5A-5J depict information and data relating to the characterizationand optimization of CHOMP components of some embodiments. FIG. 5A, Threerepresentative log-log flow cytometry scatter plots showingautofluorescence as well as reporter co-transfected with and withoutTEVP. Citrine signal is represented on the y-axis and theco-transfection marker mCherry on the x-axis. Dashed lines indicate thegate on mCherry expression analyzed in FIG. 1E. The histograms and datapoints are the same as in FIG. 1E, except for the additionally displayedautofluorescence distribution. FIG. 5B, Dose-response curves foractivatable (left) and repressible (right) TEVP reporters (indicatedschematically above each plot). The solid lines are fits based on thesame equations as those used in bandpass analysis. FIG. 5C, FIG. 5D,Reporters activatable (left) and repressible (right) by TVMVP (FIG. 5C)and HCVP (FIG. 5D). The designs are identical to those of the TEVPreporters with two exceptions: First, the specific cleavage sitesequences have been replaced with those of the regulatory protease.Second, the repressible HCVP reporter contains an additional leucinezipper compared to the other constructs, and it exhibits strongerrepression when HCVP is tagged with the complementary leucine zipper(both shown in schematic, right-hand side of (FIG. 5D). FIG. 5E,Incorporating a leucine zipper (zig-zag) on HCVP (left) enhancesrepression of TEVP but has minimal effects when used on TVMVP (right).FIG. 5F, Alignment of TEVP and TVMVP sequences enables identification ofTVMVP split site (vertical bars). FIG. 5G, A similar design enablesrepression of split TVMVP by TEVP. FIG. 5H, TVMVP can repress asingle-chain TEVP. FIG. 5I, The single-chain TVMVP is repressed by HCVP(left) and TEVP (right). FIG. 5J, An alternative three protease cascade,distinct from that in FIG. 1J, can also propagate signals.

FIGS. 5K-5R depict non-limiting examples of target proteins andsynthetic protein circuits in accordance with some embodiments.

FIGS. 6A-6C depict expanded schematics for examples of logic gates andcharacterization of examples of OR, AND, and NOR logic gates. FIG. 6A,Expanded schematic diagrams of logic gates for each input state. Foreach gate, the corresponding diagram is shown on top, followed by theexpected behavior in each of the four input states, with or without TEVPand HCVP. The presence of Citrine indicates the “ON” output state, whiledegraded Citrine (shown as chopped up reporter) represents the “OFF”state. FIG. 6B, Responses of logic gates across 16 input concentrationcombinations for OR, AND, and NOR gates. Fluorescent intensities werenormalized to the corresponding reporter stabilized with TMP (OR andNOR) or TMP and SHIELD1 (AND). In each case, reporter was used at aconcentration of 150 ng. FIG. 6C, Varying reporter expression levels bytransfecting OR, AND, and NOR reporter plasmids at 30 ng and 150 ng.Left axis displays fluorescent intensity values normalized to reporterstabilized with TMP or TMP and SHIELD1 Inputs TEVP and HCVP at 150 ngeach. Right axis shows raw fluorescent intensity values.

FIG. 6D depicts non-limiting examples of compound proteases, targetproteins, and synthetic protein circuits in accordance with someembodiments.

FIGS. 7A and 7B show information relating to expanding the inputs andcomplexity of logic gates in accordance with some embodiments. FIG. 7A,Characterization of OR, AND, and NOR gates using small molecule inputs.Asunaprevir (ASV), an inhibitor of HCVP and rapamycin, a chemicalinducer of dimerization of a FRB/FKBP and thereby an inducer of splitTEVP, were used as inputs. Each plot shows the output behavior in thepresence or absence of each of the two small molecule inputs. Theexpected presence or absence of input protease activities is shown belowthe inducer rows. FIG. 7B, NOR gates can be composed. Left, diagram ofnested NOR gate. In this example, soybean mosaic virus protease (SMVP)and herpes simplex virus Protease (HSVP) are inputs to HCVP activity.HCVP and TEVP are, in turn, inputs to TVMVP. Finally, TVMVP stabilizesthe Citrine reporter. Right, performance of the nested NOR gate withprotease inputs SMBVP, HSVP, and TEVP indicated in graph. SMVP at 80 ng,HSVP at 150 ng, TEVP at 30 ng, HCVP at 100 ng, and TVMVP at 100 ng.

FIGS. 8A-8D show information relating to characterization of bandpassand pulse-generation circuits in accordance with some embodiments. FIG.8A, Linear correlation between the amount of transfected DNA and Citrineexpression from CMV promoter. FIG. 8B, Bandpass behavior in response toTEVP and TVMVP expressed at constant DNA concentration but withdifferent levels of induction by tetracycline analog 4-epi-Tc, x-axis).FIG. 8C, A TEVP variant activated by rapamycin-mediated dimerization ofFKBP and FRB domains exhibits rapamycin-dependent activation. FIG. 8D,Left, diagram for activation of the IFP reporter by TEVP cleavage.Right, flow cytometry analysis of the dynamics of the pulse generationcircuit (also see FIGS. 3E and 3F for diagrams). Each dot represents themode of the reporter fluorescence distribution at each time point. Thesedata were obtained with the same stable cell line as in FIG. 3H.

FIG. 8E is a plot showing pulse flow data in accordance with someembodiments.

FIGS. 9A-9G show information relating to characterization andoptimization of circuits that selectively activate Casp3 in response toRas activation in accordance with some embodiments. FIG. 9A, Expandedschematic diagram of the full circuit and each of its regulatoryinteractions (numbered arrows and corresponding boxes). FIG. 9B, Exampleof reduction index analysis. The reduction index is calculated bycomparing the number of surviving transfected cells in experimental vs.Citrine-only conditions, normalized to their respective untransfectedpopulations, as shown in the equation. Dashed lines indicate individualGaussian distributions in the two-component fit, and their sum. FIG. 9C,Response of RasTEVP to physiological ligand epidermal growth factor,EGF. Left, diagram for activation of the membrane-localized IFP reporter(same as iTEV used in the pulse circuit (FIG. 3E) but with an additional12 amino-acid N-terminal signal peptide from Lyn for membranelocalization) by RasTEVP cleavage upon EGF stimulation. Right,co-transfection of iTEV reporter and RasTEVP or constitutively dimerizedmembrane-localized TEVP (‘neg ctrl TEVP’). Left two bars show RasTEVPactivation in response to EGF. Right two bars show negative controlTEVP's relatively lower response to EGF stimulation. These transfectionsincluded 25 ng of RasTEVP and 5 ng each for the negative control TEVPcomponents. EGF was used at 25 ng/mL. FIG. 9D, CytoplasmicTEVP-activatable Casp3 causes limited reduction of cell number in thepresence of membrane-localized TEVP reconstituted through leucinezippers (compare to FIG. 4B). FIG. 9E, Reduction index is unaffected bySOS_(CA) status in the presence of constitutive Casp3 activation with noRas-dependent regulation (Casp3 not depicted). For the left bars, TEVPis constitutively active through the membrane-tethered leucine zippers.The right bars uses a G12V mutation in Ras that renders itconstitutively active. FIG. 9F, The effects of RasTEVP and Casp3 doseson reduction index. Each bar represents the reduction indices fromindicated concentrations of RasTEVP and Casp3 plasmids in control orSOS_(CA) cells. FIG. 9G, Dose of TVMVP tunes the circuit's selectivityfor SOS_(CA) cells (the first and fourth pairs of bars also shown inFIG. 4C). 90 ng of RasTEVP and Casp3 were transfected in each case.

FIGS. 9H and 9I are plots showing titration data in accordance with someembodiments.

FIGS. 10A-10D show information relating to characterization andoptimization of example circuits that selectively activate Casp3 inresponse to Ras activation in accordance with some embodiments. FIG.10A, Analysis of contributions of individual regulatory edges in FIG. 9Ato overall selectivity. Left, removing TVMVP┤Casp3 (Arm 3) increasesreduction index for both control and SOS_(CA) cells; middle, removingRasTEVP┤TVMVP (Arm 4) decreases reduction in SOS_(CA) cells; right,removing TVMVP┤RasTEVP (Arm 5) has no significant effect. Despite thequalitatively consistent selectivity, there is quantitative day-to-dayvariability. FIG. 10B, IRES variants with reported strengths of 30% and70% of wild-type strength can be used to optimize TVMVP expression levelin a single transcript. The IRES variant reported to express at ˜70%level of wild type balances survival of control cells and reduction ofSOS_(CA) cells. 200 ng for each single-transcript variant. FIG. 10C,Optimizing transfection dose for full single-transcript circuit with 70%IRES. Each pair of bars represents 4 replicate co-cultures (gray dots)of control and SOS_(CA) cells transfected with the indicated amount ofthe single-transcript circuit. FIG. 10D, Annexin V staining of control,SOS_(CA) and EGFRvIII+ cells. Transfection of a negative control, fullcircuit and the positive control circuit from FIG. 4D into each cellline at 50 ng each. The fraction of apoptotic cells in all conditionswas smaller than what would be indicated by reduction index, as expecteddue to heterogeneity in the timing of initiation of apoptosis and theloss of Annexin-V+ cells due to cell death. The two effects togethercaused any given time window to capture only a fraction of thecumulative number of Annexin-V+ cells over the whole time-course.

FIG. 10E is a plot showing topology comparison data in accordance withsome embodiments.

FIGS. 10F and 10G are plots showing titration data in accordance withsome embodiments.

FIG. 11 show information relating to simulated protease-protease andTF-TF regulation dynamics in accordance with some embodiments. This plotcompares the dynamic response of some embodiments of protease-proteaseregulation and transcriptional regulation to step changes in an inputprotease/TF.

FIGS. 12A-12D depict designs and resulting data of example compoundproteases in accordance with some embodiments described herein.

FIGS. 13A-13D depict information relating to the design of non-limitingexample composable activatable protein components. FIG. 13A, Design forprotease-activatable proteases. TVMVP is expressed as a single-chainsplit variant with dimerizing leucine zippers. As indicated the cagedTVMVP has an active N-terminal half (nTVMVP) and inactive C-terminalhalf (cTVMVP). A leucine-zipper tagged active cTVMVP is co-expressed. Anactive TEVP can cleave the caging inactive cTVMVP away, allowing activenTVMVP to dimerize with active cTVMVP, effectively activating TVMVP.FIG. 13B, The same design can be applied to TEVP activated by TVMVP. B,A new tripartite split HCVP where the active HCVP lobe is split in half.The N-terminal half is co-expressed with the activating co-peptide.Dimerization of the two halves with leucine zippers reconstitutesactivity. FIG. 13C, Activatable HCVP by TEVP. FIG. 13D, Comparisonbetween the same single-caged design applied to TEVP and a double-cageddesign in which both halves of the TEVP are caged by inactive domains.

FIGS. 14A-14C depict information relating to a non-limiting example ofan intein zymogen design to activate proteases. FIG. 14A, Inteinzymogens along with ‘caging’ exteins of inactive protease halvesdecreases basal splicing. FIG. 14B, Leucine zippers along with theextein inactive protease cage and intein zymogen (ZEI-cage) enhanceprotease activation. FIG. 14C, ZEI-cage is modular and can activateorthogonal intein pairs, NrdJ1, GP41-1, and Npu, as shown with NrdJ1TEVP, GP41-1 HCVP, and Npu TVMVP.

DETAILED DESCRIPTION

Some embodiments of the systems, methods and compositions describedherein relate to a compound protease. In some embodiments, the compoundprotease comprises a protease domain with a cut site for anotherprotease, wherein the compound protease is deactivated by cleavage ofcut site for the other protease. In some embodiments, the compoundprotease is activated or deactivated by another protease, therebyforming a protein circuit. The protein circuits may be programmable withdifferent variations on the proteases and their targets to, for example,perform logic gate functions, or be part of bandpass or adaptive pulsecircuits. Applications include use in kill switches, synthetic circuits,therapeutics, gene drive payloads, cell fate control, extracellularprotein circuits such as those that control clotting, and subcellularfunctions.

Described herein are methods, compositions, and systems for engineeringviral proteases to regulate one another and/or target proteins. It isherein shown that the methods enable engineering of circuits thatperform regulatory cascades, binary logic computations, analog band-passsignal processing, generation of dynamic behaviors such as pulsing,coupling to endogenous cellular states such as oncogene activation,and/or the ability to control cellular behaviors such as apoptosis. Theflexibility and scalability of the system enables it to be reconfiguredto implement a broad range of additional functions in some embodiments.The circuits can also be encoded and delivered to cells in multipleformats, including DNA, RNA, and at the protein level itself, enablingversatile applications with or without genomic integration ormutagenesis.

Some non-limiting examples of compound proteases are shown in FIGS.1A-1C In some embodiments, the compound protease 520 comprises a) aprotease domain 520 a comprising: a first part 528 of the proteasedomain 520 a, and a second part 529 of the protease domain 520 a,wherein when the first part 528 and the second part 529 of the proteasedomain 520 a are associated together, they form an active proteasedomain 520 a, and wherein the first part 528 and the second part 529 ofthe protease domain 520 a do not self-associate on their own to form theactive protease domain 520 a; b) a cut site 515, wherein the cut site515 comprises: a first part 514 of the cut site 515, wherein the firstpart 514 of the cut site 515 is linked to the first part 528 of theprotease domain 520 a; and a second part 516 of the cut site 515,wherein the second part 516 of the cut site 515 is linked to the secondpart 529 of the protease domain 520 a, wherein when the first and secondparts 514, 516 of the cut site 515 are associated together they form anactive cut site 515 for an enzyme, and wherein when the active cut site515 is cut by the enzyme, the first and second parts 514, 516 of the cutsite 515 dissociate from one another; and c) an association domain 558,the association domain 558 comprising: a first part 557 of theassociation domain 558 that is conjugated to the second part 516 of thecut site 515; a second part 559 of the association domain 558 that islinked to the second part 529 of the protease domain 520 a, wherein theassociation domain 558 is configured to stabilize the active proteasedomain 520 a.

As described herein, a “compound protease” refers to a protease with atleast two parts of a protease domain. The parts may be linked togetherby one or more cut sites such as a cut site specific for anotherprotease. The parts of the protease domain may but need not be separatesubunits of the protease, or may include separate portions of a peptideor peptides that makes up the protease.

As described to herein, a “protease domain” includes one or morepeptides that when associated together have protease activity. Forexample, the protease activity may be the ability to cleave anotherpeptide.

As described herein, a “cut site” is a peptide sequence specific for oneor more proteases that when recognized or bound by the one or moreproteases are cleaved by the one or more proteases. The peptide sequenceof the cut site may be specific for one protease or a type of proteases,or may be general to multiple proteases or types of proteases.

As used herein, “linked” or “connected” may mean directly or indirectlylinked or connected. A non-limiting example of a direct link orconnection includes a covalent bond such as a peptide, amino, amide, orphosphodiester bond. Another non-limiting example of a direct link orconnection includes a noncovalent bond such as a hydrogen bond, ahydrophobic bond, or a hydrophilic bond. A non-limiting example of anindirect link or connection between two molecules is a covalent ornoncovalent bond between each of the two molecules but where the bond isto a third molecule (such as an association domain) that binds to eachof the two molecules.

As used herein, “stabilize” may refer to the ability of a peptide ormolecule to maintain the same or another molecule or peptide in aparticular state such as an active conformation. “Stabilize” may alsorefer to the ability of a peptide or molecule to prevent or decrease theamount of degradation that the same or another molecule or peptidefaces.

As used herein, “destabilize” may refer to the ability of a peptide ormolecule to prevent or stop the same or another molecule or peptide frommaintaining a particular state. “Destabilize” may also refer to theability of a peptide or molecule to allow or increase the amount ofdegradation that the same or another molecule or peptide faces, such asby increasing the affinity of the same or other molecule or peptide to adigestive protein.

Some embodiments include the use of degrons. Examples of degrons includea portion of a protein that affect the regulation of protein degradationrates. Some degrons are ubiquitin-dependent or ubiquitin-independent.

Some embodiments of the compound protease include a protease domain.Examples of protease domains are shown in FIGS. 1A and 1B. The proteasedomain 520A in each of FIGS. 1A and 1B includes a first part 528 and asecond part 529. Examples of a first and second part of a proteasedomain include separate halves or pieces of a dimer that work togetherto cleave a peptide, or separate portions of a protease that do notdimerize or that are not halves. For example, one part of a proteasedomain may be a fourth of the protease while another part of theprotease domain may be three fourths of the protease domain, or theremay be more than two parts. Each of the parts 528, 529 of each of theprotease domains 520 a in FIGS. 1A and 1B are separate halves of theprotease domain 520 a and are connected to a cut site 515, 515 b by alinking peptide 513, 518. Another example of a protease domain 520 a isshown in FIG. 1C, which includes a first part 528 and a second part 529,wherein the first part 528 is larger than the second part 529. In theexample in FIG. 1C, the first and second parts 528, 529 of the proteasedomain 520 a are each connected to different parts 514, 516 of a singlecut site 515 by linking peptides 513, 517.

In some embodiments, the protease domain comprises a first part 528 ofthe protease domain 520 a, and a second part 529 of the protease domain520 a. In some embodiments, the first part 528 and the second part 529of the protease domain 520 a associate together. In some embodiments,when the first part 528 and the second part 529 of the protease domain520 a are associated together, they form an active protease domain 520a. In some embodiments, the first part 528 and the second part 529 ofthe protease domain 520 a do not self-associate on their own to form theactive protease domain 520 a. For example, the protease domain 520 a mayinclude a first part 528 of the protease domain 520 a, and a second part529 of the protease domain 520 a, wherein when the first part 528 andthe second part 529 of the protease domain 520 a are associatedtogether, they form an active protease domain 520 a, and wherein thefirst part 528 and the second part 529 of the protease domain 520 a donot self-associate on their own to form the active protease domain 520a.

Some embodiments of the compound protease include a cut site. A cut sitemay be made of two parts that associate together to form the cut site.The cut site may be specific to an individual protease, or may bespecific to multiple proteases. Examples of cut sites are shown in FIGS.1A-1C. In the examples shown in each of FIGS. 1A and 1B, two cut sites515, 515 b are shown. One of the cut sites in each of FIGS. 1A and 1B515 includes a first part 514 of the cut site 515 and a second part 516of the cut site 515, the first part 514 connecting to the first part 528of the protease domain 520, and the second part 516 of the cut site 515connecting directly to a part 557 of an association domain 558 andlinking indirectly through the association domain 558 to the second part529 of the protease domain 520 a. The second site 515 b in each of FIGS.1A and 1B also includes a first part 514 b of the cut site 515 b and asecond part 516 b of the cut site 515 b, the first part 514 b connectingdirectly to the second part 529 of the protease domain 520 a, and thesecond part 516 b of the cut site 515 b connecting directly to a part559 of the association domain 558 and linking indirectly through theassociation domain 558 to the first part 528 of the protease domain 520.In the example shown in FIG. 1C, the protease 520 includes a single cut515 having two parts 514, 516, each part connecting directly to a part528, 529 of the protease domain 520 a through a linking peptide 513,517.

In some embodiments, the cut site comprises a first part 514 of the cutsite 515. In some embodiments, the first part 514 of the cut site 515 islinked to the first part 528 of the protease domain 520 a. In someembodiments, the cut site comprises a second part 516 of the cut site515. In some embodiments, the second part 516 of the cut site 515 islinked to the second part 529 of the protease domain 520 a. In someembodiments, the first and second parts 514, 516 of the cut site 515associate together. In some embodiments, when the first and second parts514, 516 of the cut site 515 are associated together they form an activecut site 515 for an enzyme. In some embodiments, when the active cutsite 515 is cut by the enzyme, the first and second parts 514, 516 ofthe cut site 515 dissociate from one another. In some embodiments, whenthe first and second parts 514, 516 of the cut site 515 are dissociatedfrom one another, the protease domain 520 a is inactive or deactivated.For example, the cut site may include a first part 514 of the cut site515, wherein the first part 514 of the cut site 515 is linked to thefirst part 528 of the protease domain 520 a; and a second part 516 ofthe cut site 515, wherein the second part 516 of the cut site 515 islinked to the second part 529 of the protease domain 520 a, wherein whenthe first and second parts 514, 516 of the cut site 515 are associatedtogether they form an active cut site 515 for an enzyme, and whereinwhen the active cut site 515 is cut by the enzyme, the first and secondparts 514, 516 of the cut site 515 dissociate from one another.

Some embodiments of the compound protease include an association domain.An example of an association domain is shown in FIG. 1A. The associationdomain 558 in FIG. 1A includes two parts 557, 559 each binding togethernoncovalently to ultimately link the first and second parts 528, 529 ofthe protease domain 520 a together. Another example of an associationdomain is shown in FIG. 1B. The association domain 558 in FIG. 1Bincludes a single peptide strand with two parts 557, 559 that eachconnect to a cut site 515, 515 b and ultimately link the first andsecond parts 528, 529 of the protease domain 520 a together. In someembodiments, the association domain comprises a first part 557 of theassociation domain 558. In some embodiments, the first part 557 of theassociation domain 558 is conjugated to the second part 516 of the cutsite 515. In some embodiments, the association domain comprises a secondpart 559 of the association domain 558. In some embodiments, the secondpart 559 of the association domain 558 is linked to the second part 529of the protease domain 520 a. In some embodiments, the associationdomain 558 is configured to stabilize the active protease domain 520 a.For example, the association domain may include a first part 557 of theassociation domain 558 that is conjugated to the second part 516 of thecut site 515; a second part 559 of the association domain 558 that islinked to the second part 529 of the protease domain 520 a, wherein theassociation domain 558 is configured to stabilize the active proteasedomain 520 a.

Examples of association domains include a leucine zipper motif or acomplementary leucine zipper motif, a scaffold protein or a fragmentthereof, a scaffold-binding motif, an antibody, an epitope,tetratricopeptide repeat, a tetracopeptide repeat-binding motif, aG-protein-coupled receptor, a β-arrestin, and/or a G protein. In someembodiments, the association domain includes any protein(s) orcomponent(s) of protein(s) that bind together. Thus, the associationdomain is contemplated to cover any protein:protein interactionaccording to some embodiments. In some embodiments, the associationdomain includes a ligand-binding protein or domain and/or the ligand.

In some embodiments of the compound protease, the first and second partsof the association domain of the compound protease comprise separatepeptide strands that hybridize together, for example, as shown in FIG.1A. In some embodiments of the compound protease, the first and secondparts 557, 559 of the association domain 558 of the compound protease520 are a single peptide strand, for example, as shown in FIG. 1B.

Some embodiments do not include an association domain linking the firstand second parts 528, 529 of a protease domain 520 a together. Forexample, in the example shown in FIG. 1C, the first and second parts528, 529 of the protease domain 520 a are instead linked togetherthrough the cut site. The example in FIG. 1C shows the use of optionallinking peptides 513, 517 which some embodiments do not include. Theexample in FIG. 1C does include a part 556 of an association domain fora different purpose—that of helping to recruit another protease orcompound protease to the cut site 515 of the protease 520 in FIG. 1C.For example, the other protease or compound protease may be recruited tothe cut site 515 of the protease 520 in FIG. 1C when the other proteaseor compound protease includes a complementary part of the associationdomain to the part 556 of the association domain included on theprotease domain 520 a of the protease 520 shown in FIG. 1C.

In some embodiments, the compound protease comprises or consists of atobacco etch virus NIa (TEV) protease, tobacco vein mottling virus(TVMV) NIa protease, sugarcane mosaic virus NIa protease, sunflower mildmosaic virus NIa protease, turnip mosaic virus NIa protease, plum poxvirus NIa protease, soybean mosaic virus protease, hepatitis c virus(HCV) ns3 protease, hepatitis a virus 3c protease, dengue virus NS3protease, zika virus NS3 protease, yellow fever virus NS3 protease, orhuman herpes virus 1 protease. In some embodiments, the compoundprotease comprises or consists of a human site-specific protease such asthrombin and/or enteropeptidase.

Some embodiments comprise or consist of a nucleic acid encoding thecompound protease. Examples of nucleic acids include DNA and RNA.

Embodiments

Proteases

Some embodiments of the compounds, methods or systems described hereinrelate to a protease such as a compound protease. In some embodiments,the protease includes any protease as described herein. For example, theprotease may include a protease as described under any of thesubheadings, “Proteases,” “Systems,” and/or “Methods.”

In some embodiments, the compound protease includes a protease domain,one or more cut sites, and/or one or more association domains and/orparts of association domains. In some embodiments, the protease includesa compound protease such as is shown in any of FIGS. 1A-1C. For example,the protease may include a) a protease domain 520 a including: a firstpart 528 of the protease domain 520 a, and a second part 529 of theprotease domain 520 a, wherein when the first part 528 and the secondpart 529 of the protease domain 520 a are associated together, they forman active protease domain 520 a, and wherein the first part 528 and thesecond part 529 of the protease domain 520 a do not self-associate ontheir own at physiological conditions to form the active protease domain520 a; b) a cut site 515, wherein the cut site 515 includes: a firstpart 514 of the cut site 515, wherein the first part 514 of the cut site515 is linked to the first part 528 of the protease domain 520 a; and asecond part 516 of the cut site 515, wherein the second part 516 of thecut site 515 is linked to the second part 529 of the protease domain 520a, wherein when the first and second parts 514, 516 of the cut site 515are associated together they form an active cut site 515 for an enzyme,and wherein when the active cut site 515 is cut by the enzyme, the firstand second parts 514, 516 of the cut site 515 dissociate from oneanother; and c) an association domain 558, the association domain 558including: a first part 557 of the association domain 558 that isconjugated to the second part 516 of the cut site 515; a second part 559of the association domain 558 that is linked to the second part 529 ofthe protease domain 520 a, wherein the association domain 558 isconfigured to stabilize the active protease domain 520 a.

In some embodiments, the ability or lack thereof of the first part 528and the second part 529 of the protease domain 520 a to self-associateon their own to form the active protease domain 520 a is concentrationdependent such that at physiological conditions they do notself-associate.

In some embodiments, the protease domain comprises, is comprised of, oris composed of a peptide or co-peptide, or multiple peptides orco-peptides.

In some embodiments, the compound protease includes one or more cutsites. In some embodiments, one or more of the cut sites are specificfor a different protease or different proteases than the compoundprotease. For example, the compound protease would not be able to cleaveitself according to some embodiments. Thus, in some embodiments, thecompound protease is not naturally occurring, and/or the compoundprotease does not include a natural cut site (such as for the proteaseitself). For example, the compound protease may not include a naturalcut site for itself between a main protease domain and a co-peptide ofthe compound protease.

Some embodiments of the protease include a compound protease such as thecompound protease 520 shown in FIG. 1C, the compound protease 520including: a) a protease domain 520 a including: a first part 528 of theprotease domain 520 a, and a second part 529 of the protease domain 520a, wherein when the first part 528 and the second part 529 of theprotease domain 520 a are associated together, they form an activeprotease domain 520 a, and wherein the first part 528 and the secondpart 529 of the protease domain 520 a do not self-associate on their ownto form the active protease domain 520 a; b) a cut site 515, wherein thecut site 515 includes: a first part 514 of the cut site 515, wherein thefirst part 514 of the cut site 515 is linked to the first part 528 ofthe protease domain 520 a; and a second part 516 of the cut site 515,wherein the second part 516 of the cut site 515 is linked to the secondpart 529 of the protease domain 520 a, wherein when the first and secondparts 514, 516 of the cut site 515 are associated together they form anactive cut site 515 for an enzyme, and wherein when the active cut site515 is cut by the enzyme, the first and second parts 514, 516 of the cutsite 515 dissociate from one another; c) a first peptide 513 connectingthe first part 528 of the protease domain 520 a to the first part 514 ofthe cut site 515; and d) a second peptide 517 connecting the second part529 of the protease domain 520 a to the second part 516 of the cut site515, wherein the first and second linkers 513, 517 are configured tostabilize the active protease domain 520 a. In some embodiments, thefirst peptide 513 connecting the first part 528 of the protease domain520 a to the first part 514 of the cut site 515 includes a linker. Insome embodiments, the second peptide 517 connecting the second part 529of the protease domain 520 a to the second part 516 of the cut site 515includes a linker.

Some embodiments of the protease include a compound protease such as thecompound protease 520 shown in FIGS. 1A-1C, the compound protease 520including: a) a protease domain 520 a including: a first part 528 of theprotease domain 520 a, and a second part 529 of the protease domain 520a, wherein when the first 528 part and the second part 529 of theprotease domain 520 a are associated together, they form an activeprotease, and wherein the first part 528 and the second part 529 of theprotease domain 520 a do not self-associate on their own to form theactive protease; and b) a cut site 515, wherein the cut site 515includes: a first part 514 of the cut site 515, wherein the first part514 of the cut site 515 is linked to the first part 528 of the proteasedomain 520 a; and a second part 516 of the cut site 515, wherein thesecond part 516 of the cut site 515 is linked or indirectly connected tothe second part 529 of the protease domain 520 a, wherein when the firstand second parts 514, 516 of the cut site 515 are associated togetherthey form an active cut site 515 for an enzyme, and wherein when theactive cut site 515 is cut by the enzyme, the first and second parts514, 516 of the cut site dissociate from one another.

In some embodiments of the compound protease, such as is shown in FIG.1C, the first part 514 of the cut site 515 is covalently linked to thefirst part 528 of the protease domain 520 a by a first peptide linkage513, and/or wherein the second part 516 of the cut site 515 iscovalently linked to the second part 529 of the protease domain 520 a bya second peptide linkage 517.

Some embodiments of the proteases described herein include one or morelinkers or linker peptides. The linkers or linker peptides may connector link (directly or indirectly, and/or covalently or noncovalently)various parts of the protease such as a cut site or a part of the cutsite to a protease domain or a part of a protease domain. However, thisdisclosure is not limited to only linkers or linker peptides connectingthe protease parts. Examples of a linker is a peptide that includes1-10, 10-25, 25-50, 50-100, or 100-1000 amino acids. For example, thecompound protease may include a first peptide linkage 513 that includesa linker peptide including 1-10, 10-25, 25-50, 50-100, or 100-1000 aminoacids, and/or a second peptide linkage 517 includes a linker peptideincluding 1-10, 10-25, 25-50, 50-100, or 100-1000 amino acids.

In some embodiments of the compound protease, wherein the second part529 of the protease domain 520 a includes a part 556 of an associationdomain connected to the second part 529 of the protease domain 520 a,wherein the part 556 of the association domain connected to the secondpart 529 of the protease domain 520 a is configured to recruit theenzyme to the active cut site 515 by binding a second part of theassociation domain on the enzyme.

In some embodiments of the compound protease, such as is shown in FIGS.1A and 1B, the compound protease includes a second cut site 515 b,wherein the second cut site 515 b includes: a first part 514 b of thesecond cut site 515 b, wherein the first part 514 b of the second cutsite 515 b is linked to the second part 529 of the protease domain 520a; and a second part 516 b of the second cut site 515 b, wherein thesecond part 516 b of the second cut site 515 b is linked or indirectlyconnected to the first part 528 of the protease domain 520 a; whereinwhen the first and second parts 514 b, 516 b of the second cut site 515b are associated together they form an active second cut site 515 b forthe enzyme, and wherein when the active second cut site 515 b is cut bythe enzyme, the first and second parts 514 b, 516 b of the second cutsite dissociate from one another.

In some embodiments of the compound protease, such as is shown in FIG.1A, the compound protease includes an association domain 558 thatincludes: a first part 557 of the association domain 558, conjugated tothe second part 516 of the first cut site 515; a second part 559 of theassociation domain 558, conjugated to the second part 516 b of thesecond cut site 515 b, wherein the association domain 558 is configureto stabilize the active protease domain. In some embodiments of thecompound protease, the first part 514 of the cut site 515 is covalentlylinked to the first part 528 of the protease domain 520 a by a firstpeptide linkage 513, and/or wherein the first part 514 b of the secondcut site 515 b is covalently linked to the second part 529 of theprotease domain 520 a by a second peptide linkage 518. In someembodiments, the first peptide linkage 513 includes a linker peptideincluding 1-10, 10-25, 25-50, 50-100, or 100-1000 amino acids. In someembodiments, the second peptide linkage 518 includes a linker peptideincluding 1-10, 10-25, 25-50, 50-100, or 100-1000 amino acids. In someembodiments of the compound protease, the second part 516 of the cutsite 515 is indirectly connected to the second part 529 of the proteasedomain 520 a through the association domain 558, wherein the first andsecond parts 557, 559 of the association domain are covalently ornon-covalently linked together.

Some embodiments of the compound protease, such as the example shown inFIG. 1B, include an association domain 558 of the compound protease 520including a first part 557 and a second part 559, wherein the first part557 of the association domain 558 links to the second part 516 of thefirst cut site 515, and wherein the second part 559 of the associationdomain 558 links to the second part 516 b of the second cut site 515 b.In some embodiments, the first part 514 of the cut site 515 iscovalently linked to the first part 528 of the protease domain 520 a bya first peptide linkage 513, and/or wherein the first part 514 b of thesecond cut site 515 b is covalently linked to the second part 529 of theprotease domain 520 a by a second peptide linkage 518. In someembodiments, the first peptide linkage 513 includes a linker peptideincluding 1-10, 10-25, 25-50, 50-100, or 100-1000 amino acids. In someembodiments, the second peptide linkage 518 includes a linker peptideincluding 1-10, 10-25, 25-50, 50-100, or 100-1000 amino acids. In someembodiments, the second part 516 of the cut site 515 is indirectlyconnected to the second part 529 of the protease domain 520 a throughthe association domain 558. In some embodiments, the association domainconnecting to the second part 516 of the cut site 515 and to the to thesecond part 516 b of the second cut site 515 b is configured to recruitthe enzyme to the active cut site 515 and/or to the active second cutsite 515 b by binding a second part of the association domain on theenzyme.

In some embodiments of the compound protease, the compound proteaseincludes a degron. In some embodiments, the compound protease includesmultiple degrons. In some embodiments, at lease one degron of thecompound protease destabilizes the compound protease when present on thecompound protease by enhancing degradation of the compound protease. Insome embodiments, at least one of the degrons of the compound proteaseis or comprises a conditional N-end degron. In some such embodiments,the at least one degron or the condition N-end degron does notinactivate or destabilize the compound protease until the degron or acomponent thereof is cleaved by another protease to reveal the degronand allow it to stabilize the compound protease. In some embodiments,one or more degrons of the compound protease comprise a conditionalN-end degron such as an N-end degron that is conditional on cleavage ofa cut site specific for an enzyme, a second protease, or the compoundprotease, on the compound protease.

In some embodiments, the protease or compound protease is a viralprotease, or is a modified form of a viral protease. In someembodiments, the protease or compound protease is a mammalian or humanprotease, or is a modified form of a mammalian or human protease.

Some embodiments of the compound proteases or of a target protein for aprotease include a localization tag. For example, the protease 120 shownin the example in FIG. 4A (panel 3) includes a membrane targeting signal(mts). Such localization enable sub-cellular computation or signaltransduction in some embodiments. In some cases, the protease (or splitprotease) includes the localization sequence at one or more termini ofprotease.

Some embodiments relate to a protease such as a compound protease thatinteracts with another enzyme or protease by being positively regulatedby that other enzyme or protease. As shown in FIG. 12C, the inventorsobserved some positive regulatory effect in a design where one proteaseclips a degron off of another protease. Thus, some embodiments of thecompound protease include a degron linked to a protease domain or othercomponent of the compound protease by a cut site of the compoundprotease. The degron may act to destabilize the compound protease aslong as the degron is present on the compound protease. In some suchembodiments, cleavage of the cut site removes the degron to stabilizethe compound protease. In some embodiments, the compound protease isconfigured to be activated and/or destabilized by another compoundprotease, protease, or enzyme. In some embodiments, the compoundprotease is configured to be deactivated and/or destabilized by anothercompound protease, protease, or enzyme.

Some embodiments relate to positive regulation for cellularprotein-level regulation circuits. Positive regulation of one proteinactivity by another is beneficial for some protein-level circuits. Hereis described some designs and experimental results establishing theability to achieve positive protein-protein regulation in a modularfashion. Two classes of designs are focused on here, but otherembodiments are envisioned: (1) Reversible activation by swappableassociation domains; (2) Irreversible activation by intein-mediatedprotein splicing.

In some embodiments, the compound protease is cleavage-activatable byanother protease. For example, the compound protease may be tagged withan auto-inhibitory domain that can be removed with another protease(FIG. 12A). In some embodiments, the compound protease is tagged with adegron (such as a DHFR degron) that can be removed with another protease(FIG. 12B). In some embodiments, the compound protease comprises a splitprotease tagged with a degron (for example, four tandem repeats ofubiquitin) on the end of a leucine zipper, and the degron is removableby another protease (FIG. 12C). In some embodiments, the compoundprotease includes an N-terminal half that is caged with a complementaryleucine zipper and/or a catalytically inactive C-terminal half, and thecaging domains are removable with another protease (FIG. 12D).

Some embodiments relate to a compound protease, the compound proteasecomprising: a) a protease domain comprising: a first part of theprotease domain, and a second part of the protease domain, wherein whenthe first part and the second part of the protease domain are associatedtogether, they form an active protease domain; and/or b) a cut site,wherein the cut site comprises: a first part of the cut site, and asecond part of the cut site, wherein when the first and second parts ofthe cut site are associated together they form an active cut site for anenzyme, and wherein when the active cut site is cut by the enzyme, thefirst and second parts of the cut site dissociate from one another;wherein the compound protease is configured to be activated ordeactivated by cleavage of the active cut site by the enzyme.

Some embodiments relate to a cleavage-activatable compound protease,comprising: a) a protease domain comprising: a first part of theprotease domain, and a second part of the protease domain, wherein whenthe first part and the second part of the protease domain are associatedtogether, they form an active protease domain; and/or b) a cut site,wherein the cut site comprises: a first part of the cut site, and asecond part of the cut site, wherein when the first and second parts ofthe cut site are associated together they form an active cut site for anenzyme, and wherein when the active cut site is cut by the enzyme, thefirst and second parts of the cut site dissociate from one another;wherein the compound protease is configured to be activated by cleavageof the active cut site by the enzyme. In some embodiments, thecleavage-activatable compound protease comprises an association domain,and the association domain prevents the first part of the proteasedomain from associating with the second part of the protease until thecut site is cut by the enzyme. In some embodiments, thecleavage-activatable compound protease comprises an association domain,wherein the association domain cages the first part of the proteasedomain and prevents the first part of the protease domain fromassociating with the second part of the protease until the cut site iscut by the enzyme. In some embodiments, the cleavage-activatablecompound protease further comprises a three-way split protease.

1. Reversible activation by swappable association domains

Single Caged Design:

In this design, a target protease is ‘caged’ in an inactive form thatcan be uncaged by an activating protease to turn on its proteaseactivity. More specifically, the target protease is caged by splittingit, and including an inactivating mutation in one half (FIG. 1A).Inclusion of a cleavage site for the activating protease, allows theinactive half to be released, and permits association of the active halfwith a separately expressed active half to reconstitute an activeprotease in accordance with some embodiments.

Experimental Validation:

For an initial test, TVMVP was used as a starting protease. In asimplified design, a single-chain ‘caged’ TVMVP was expressed. The‘caged’ TVMVP comprises an active N-terminal lobe and an inactiveC-terminal lobe. Residues involved in catalytic cleavage located in theC-half of the protease domain were mutated. Heterodimerizing leucinezippers were included to maintain caging dimerization of the inactiveform. One TEVP cleavage site was inserted between the heterodimerizingleucine zippers and the inactive domain in order to allow ‘decaging’ ofthe inactive half from the active half of the protease (FIG. 13A, leftcartoon). This design was compared to a ‘caged’ TVMVP with two TEVPcleavage sites: the first one between the heterodimerizing leucinezippers and the second one between the inactive C-terminal lobe and itscorresponding leucine zipper (FIG. 13A, right cartoon). The activeC-terminal lobe of TVMVP was expressed with the correspondingheterodimerizing leucine zipper. Co-expression of the ‘caged’ TVMVP withthe active C-terminal half of TVMVP showed that this design was capableof maintaining its inactive state in the presence of the active cTVMVP.Cleavage by TEVP activated TVMVP with a single TEVP cleavage site (onesite, middle column of plot in FIG. 13A). The design with two TEVPcleavage sites (‘two sites’ in FIG. 13A) performed better in terms ofhaving an expanded dynamic range.

Double Caged Design:

The single caged design was also applied to a TEV protease (FIG. 13D,left) and enabled regulation by TVMV protease. In order to reduce thebaseline level of activity in both the ‘off’ and ‘on’ states, adouble-caged design was also developed (FIG. 13D, right). In thisdesign, two forms of the split target protease are expressed, each ofwhich contains an inactivating mutation in opposite domains, as shown.Each target protease contained cleavage sites for the activatingprotease. As a result, cleavage can enable swapping of these domains toproduce a fully active reconstituted target protease, as can be shown.

Generality:

Similar designs can be applied to additional proteases. This wasdemonstrated using a Hepatitis C Virus (HCV) protease (FIGS. 13B, 13C).First, a 3-way split HCV protease was designed. The HCVP contained acore HCVP and activity-enhancing co-peptide (small pie slice, FIG. 13B).A TEVP cleavage site was successfully inserted between the core and theactivity-enhancing peptide to successfully reduce HCVP activity (seedescription under the heading, “Further embodiments”). On top of thissplit, a second split site was added within the core HCVP domain. TheN-terminal lobe was then expressed as a single chain with itsco-peptide. Reconstitution of HCVP with heterodimerizing leucine zippershad the same level of activity as the wildtype HCVP (FIG. 13B). Thesimple uncaging design showed activation of HCV protease activity by TEVprotease (FIG. 13C).

2. Irreversible Activation by Intein-Based Activatable Proteases

Background:

Inteins, intervening proteins, are autoprocessing domains which are ableto carry out protein splicing (Gramespacher et al, JACS, 2017). Inteinsexcise themselves from a polypeptide precursor and ligate the twoexteins, external proteins, through a new peptide bond. Split inteins,unlike the contiguous inteins, are translated in two distinctpolypeptide sequences of an N-intein and C-intein, each with its ownextein. Upon association, the split inteins will perform proteinsplicing in trans. Split intein zymogens were demonstrated in which eachsplit intein pair is caged and activated upon proteolysis (Gramespacheret al, JACS, 2017).

Design and Validation:

A split intein-based activatable protease was designed, in which twohalves of a split protease are each fused to complementary cagedinteins, such that cleavage of the caged inteins can permit proteinsplicing to reconstitute the split extein as a functional protease (FIG.14A). The inventors used TEVP as a target protease and TVMVP as anactivating protease. This design showed limited activation of TEVP (FIG.14A). The inventors therefore caged the extein halves by using thecorresponding inactive protease half (FIG. 14A, protease domains). Thisdesigned successfully reduced baseline activation (FIG. 14A). Theinventors next introduced a heterodimerizing leucine zipper pair to eachhalf of the protease to sterically constrain the ability of the splitinteins to associate with each other. The inventors found that additionof the leucine zippers to our overall design enhanced spliced TEVPactivity, showing a broad dynamic range for regulation (FIG. 14B). Theinventors further demonstrated this overall design can be transferredfrom one orthogonal intein pair to another (NrdJ1, GP41-1, and Npu, FIG.14C), and these intein pairs can be used to control different outputproteases (TEVP, HCVP, and TVMVP, respectively).

Systems

Some embodiments relate to a system such as a synthetic protein circuit.The system or synthetic protein circuit may include any of the proteasesdescribed herein such as one or more of the compound proteases shown inFIGS. 1A-1C. In some embodiments, the system or synthetic proteincircuit includes a first protease, second protease, third protease,fourth protease, fifth protease, sixth protease, seventh protease,eighth protease, ninth protease and/or tenth protease. Any of saidproteases may comprise or be composed of a compound protease asdescribed herein. In some embodiments of the system or synthetic proteincircuit, the first protease 110 and the second protease 120 each includean HCV protease, a TEV protease, or a TVMV protease. Some embodimentsinclude positive protease-protease regulation (FIG. 12C). For example,some embodiments relate to a synthetic protein circuit that includes amode of positive protease-protease regulation, such as one that ismediated through degron removal.

Some embodiments relate to a synthetic protein circuit such as a proteincircuit or a part thereof shown in FIGS. 1D-1J. In some embodiments, thesynthetic protein circuit includes: a first protease 110; and a secondprotease 120 including a cut site 115 specific for the first protease110, wherein the second protease 120 is inactivated by cleavage of thecut site 115 specific for the first protease 110.

Some embodiments of the synthetic protein circuit include a targetprotein 140, such as the target protein shown in FIG. 1H, including: adegron 141 of the target protein 140 that destabilizes the targetprotein 140 when present on the target protein 140 by enhancingdegradation of the target protein 140, and a cut site 125 specific forthe second protease 120, wherein the target protein 140 is configured tobe stabilized or destabilized by cleavage of the cut site 125 specificfor the second protease 210.

In some embodiments of the synthetic protein circuit, the secondprotease 120 includes a first cleavage domain 128 and a second part 129of the cleavage domain, the first part 128 connecting to the cut site115 specific for the first protease 110, and the second part 129connecting to another cut site 115 specific for the first protease 110,the second protease's 120 two cut sites 115 specific for the firstprotease 110 each connecting to an association domain 158 of the secondprotease 120 such as a leucine zipper. In some embodiments, the secondprotease's 120 two cut sites 115 specific for the first protease 110each connect to a separate association domain 158, 159 of the secondprotease 120, wherein the second protease 120 is active when theseparate association domains 158, 159 bind together, and wherein thesecond protease 120 is configured to be deactivated by cleavage ofeither of its two cut sites 115 specific for the first protease 110. Insome embodiments, one of the second protease's 120 association domains158, 159 includes a complementary association domain 159 such as leucinezipper that is complementary or antiparallel to the other associationdomain 158 of the second protease 120. In some embodiments, such as inthe example shown in FIG. 1I, the second protease's 120 two cut sites115 specific for the first protease 110 each connect to a singleassociation domain 159 of the second protease 120, and wherein thesecond protease 120 is configured to be deactivated by cleavage ofeither of its two cut sites 115 specific for the first protease 110.

In some embodiments of the synthetic protein circuit, the first protease110 includes an association domain 158 of the first protease 110 thatbinds to a complementary association domain 159 of the second protease120, thereby allowing or enhancing the first protease's 110 ability tocleave a cut site 115 specific to the first protease 110 on the secondprotease 120.

Some embodiments of the synthetic protein circuit include a third,fourth, fifth, sixth, seventh, eighth, ninth and/or tenth protease 130,each protease 110, 120, 130 including a cut site specific to at leastone of the proteases 110, 120, 130, and wherein each protease 110, 120,130 is configured to be destabilized or deactivated by cleavage of itscut site.

Some embodiments of the synthetic protein circuit include a proteaseactivatable target protein. In some embodiments, such as in the examplesshown in FIGS. 1E and 1H, the target protein's 140 cut site 125 specificto the second protease 120 includes a first part 125 a of the cut site125 of the target protein 140 and a second part 125 b of the cut site125 of the target protein 140, the first part 125 a of the cut site 125of the target protein 140 connecting to a domain or motif 144 of thetarget protein, and the second part 125 b of the cut site 125 of thetarget protein 140 connecting to the degron 141 of the target protein140, and wherein the target protein 140 is stabilized by cleavage of itscut site 125 specific for the second protease 120.

In some embodiments of the synthetic protein circuit, such as is shownin FIG. 1F, the degron 141 of the target protein 140 includes a maskingpeptide 146 that connects to the degron 141 of the target protein 140and blocks cleavage of the target protein's 140 cut site 125 specificfor the second protease 120, wherein the masking peptide 146 of thedegron 141 of the target protein 140 includes the target protein's 140cut site 125 specific for the second protease 120, and wherein thetarget protein 140 is configured to be destabilized by cleavage of itscut site 125 specific for the second protease 120, wherein cleavage ofthe target protein's 140 cut site 125 specific for the second protease125 uncovers the target protein's 140 degron 141.

In some embodiments of the synthetic protein circuit, the target protein140 consists of or comprises a protease, a reporter protein, afluorescent protein, a scaffold, an actuator protein, a transcriptionalregulator, or a signaling protein.

In some embodiments of the system, the synthetic protein circuitincludes a logic gate such as a logic gate shown in FIGS. 2A-2I. In someembodiments, the system includes a synthetic protein circuit, including:a first protease 110, optionally including an association domain 158 ofthe first protease 110; a second protease 120, optionally including acomplementary association domain 159 of the second protease 120; and atarget protein 140 including a degron 141 of the target protein 140 thatdestabilizes the target protein 140 when present on the target protein140 by enhancing degradation of the target protein 140; wherein thetarget protein 140 is configured to interact with the first protease110, the second protease 120, a third protease 130 and/or a fourthprotease 240 to form an OR, AND, NOR, NAND, IMPLY, NIMPLY, XOR or XNORlogic gate.

In some embodiments, the synthetic protein circuit includes an OR logicgate. In some embodiments, the target protein 140 further includes a cutsite 115 specific for the first protease 110 and a cut site 125 specificfor the second protease 120 between the degron 141 of the target protein140 and a part 144 of the target protein 140, and wherein the targetprotein 140 is stabilized by cleavage of either of its cut sites 115,125.

In some embodiments, the synthetic protein circuit includes an AND logicgate. In some embodiments, the target protein 140 further includes a cutsite 115 of the target protein 140 specific for the first protease 110between the degron 141 of the target protein 140 and a part 144 of thetarget protein 140, and a cut site 125 specific for the second protease120 connected to another degron 142 of the target protein 140 and anoptional association domain 158 of the target protein 140, and whereinthe target protein 140 is stabilized by cleavage of both of its cutsites 115, 125.

In some embodiments, the synthetic protein circuit includes a NOR logicgate. In some embodiments, the synthetic protein circuit includes athird protease 130 including: a cut site 115 specific for the firstprotease 110, a cut site 125 specific for the second protease 120, andan optional association domain 158 of the third protease 130, whereinthe third protease 130 is configured to be deactivated by cleavage ofeither of its cut sites 115, 125; and wherein the target protein 140includes a cut site 135 specific for the third protease 130 between thedegron 141 of the target protein 140 and a part 144 of the targetprotein 140, wherein the target protein 140 is stabilized by cleavage ofits cut site 135 specific for the third protease 130. In someembodiments, the third protease 130 further includes a first domain 138of the third protease 130 and a second domain 139 of the third protease130; wherein the first domain 138 of the third protease 130 includes thethird protease's 130 cut sites 115, 125 specific for the first andsecond proteases 110, 120 and the optional association domain 158 of thethird protease 130; wherein the second domain 139 the third protease 130includes another cut site 115 specific for the first protease 110,another cut site 125 specific for the second protease 120, and anoptional complementary association domain 159 the third protease 130;and wherein the third protease 130 is configured to be deactivated bycleavage of any of its cut sites 115, 115, 125, 125.

In some embodiments, the synthetic protein circuit includes a NAND logicgate. In some embodiments, the synthetic protein circuit includes athird protease 130 including a cut site 115 specific for the firstprotease 110, and configured to be deactivated by cleavage of its cutsite 115; and a fourth protease 230 including a cut site 125 specificfor the second protease 120, and configured to be deactivated bycleavage of its cut site 125; wherein the target protein 140 includes acut site 135 specific for the third and fourth proteases 130, 230between the degron 141 of the target protein 140 and a part 144 of thetarget protein 140, wherein the target protein 140 is stabilized bycleavage of its cut site 135. In some embodiments, the third protease130 further includes a first domain 138 of the third protease 130, asecond domain 139 of the third protease 130, and an optionalcomplementary association domain 159 of the third protease 130; whereinthe first domain 138 of the third protease 130 includes the cut site 115specific for the first protease 110; wherein the second domain 139 ofthe third protease 130 includes another cut site 115 specific for thefirst protease 110; wherein the complementary association domain 159 thethird protease 130 optionally includes two parts 159 a, 159 b of thethird protease 130, each part 159 a, 159 b the third protease 130connected to one of the third protease's 130 cut sites 115, 115; andwherein the third protease 130 is configured to be deactivated bycleavage of either of its cut sites 115, 115.

In some embodiments of the synthetic protein circuit, the fourthprotease 230 protease further includes a first domain 238 of the fourthprotease 230, a second domain 239 of the fourth protease 230, and anoptional association domain 158 of the fourth protease 230; wherein thefirst domain 238 of the fourth protease 230 includes the cut site 125specific for the second protease 120; wherein the second domain 239 ofthe fourth protease 230 includes another cut site 125 specific for thesecond protease 120; wherein the association domain 158 of the fourthprotease 230 optionally includes two parts 158 a, 158 b, each part 158a, 158 b connected to one of the fourth protease's 230 cut sites 125,125; and wherein the fourth protease 230 is configured to be deactivatedby cleavage of either of its cut sites 125, 125.

In some embodiments, the synthetic protein circuit comprises an IMPLYlogic gate. In some embodiments, the synthetic protein circuit includesa third protease 130 including a cut site 125 specific for the secondprotease 120, and configured to be deactivated by cleavage of its cutsite 125; wherein the target protein 140 further includes a cut site 115specific for the first protease 110 and a cut site 135 specific for thethird protease 130 between the degron 141 of the target protein 140 anda part 144 of the target protein 140, and wherein the target protein 140is stabilized by cleavage of either cut sites 115, 135. In someembodiments, the third protease 130 further includes a first domain 138,a second domain 139, and an optional association domain 158; wherein thefirst domain 138 of the third protease 130 includes the third protease'scut site 125 specific for the second protease 120; wherein the seconddomain 139 of the third protease 130 includes another cut site 125specific for the second protease 120; wherein the association domain 158of the third protease 130 optionally includes two parts 158 a, 158 b ofthe third protease 130, each part 158 a, 158 b of the third protease 130connected to one of the third protease's 130 cut sites 125, 125; andwherein the third protease 130 is configured to be deactivated bycleavage of either of its cut sites 125, 125.

In some embodiments, the synthetic protein circuit comprises a NIMPLYlogic gate. In some embodiments, the synthetic protein circuit includesa third protease 130 including a cut site 115 specific for the firstprotease 110, and configured to be deactivated by cleavage of its cutsite 115; wherein the target protein 140 further includes a cut site 135specific for the third protease 130 between the degron 141 and a part144 of the target protein, and a cut site 125 specific for the secondprotease 120 connected to another degron 142 of the target protein 140and an optional association domain 158 of the target protein 140, andwherein the target protein 140 is stabilized by cleavage of both of itscut sites 125, 135. In some embodiments, the third protease 130 furtherincludes a first domain 138 of the third protease 130, a second domain139 of the third protease 130, and an optional complementary associationdomain 159 of the third protease 130; wherein the first domain 138 ofthe third protease 130 includes the cut site 115 specific for the firstprotease 110; wherein the second domain 139 of the third protease 130includes another cut site 115 specific for the first protease 110;wherein the complementary association domain 159 of the third protease130 optionally includes two parts 159 a, 159 b of the third protease130, each part 159 a, 159 b of the third protease 130 connected to oneof the third protease's 130 cut sites 115, 115; and wherein the thirdprotease 130 is configured to be deactivated by cleavage of either ofits cut sites 115, 115.

In some embodiments, the synthetic protein circuit comprises an XORlogic gate. In some embodiments, the synthetic protein circuit includesa second target 240 protein including a degron 241 of the second target240 protein that destabilizes the second target protein 240 when presenton the second target protein 240; wherein the target protein 140 furtherincludes a cut site 115 specific for the first protease 110 between itsdegron 141 and a part 144 of the target protein 140, an other degron 142of the target protein 140, and a cut site 125 specific for the secondprotease 120 connected to the other degron 142 of the target protein140, wherein the target protein 140 is destabilized by its first degron141 unless its cut site 115 specific for the first protease 110 iscleaved by the first protease 110, and wherein the target protein 140 isdestabilized by cleavage of its cut site 125 specific for the secondprotease 120; and wherein the second target protein 240 further includesa cut site 125 specific for the second protease 120 between its degron241 and the part 244 of the second target protein 240, an other degron242 of the second target protein 240, and a cut site 115 specific forthe first protease 110 connected to the other degron 242 of the secondtarget protein 240, wherein the second target protein 240 isdestabilized by its first degron 241 unless its cut site 125 specificfor the second protease 120 is cleaved by the second protease 120, andwherein the second target protein 240 is destabilized by cleavage of itscut site 115 specific for the first protease 110. In some embodiments,the second target protein 240 further includes a complementaryassociation domain 159 of the second target protein 240 connected at ornear the other degron 242 of the second target protein 240 or the secondtarget protein's 240 cut site 115 specific for the first protease 110.In some embodiments, the target protein's 140 other degron 142 includesa masking peptide 146 of the other degron 142 of the target protein 140connected to the target protein's 140 other degron 142, wherein themasking peptide 146 of the other degron 142 of the target protein 140prevents the target protein's 140 other degron 142 from destabilizingthe target protein 140 when the masking peptide 146 of the other degron142 of the target protein 140 is present on the target protein 140,wherein the masking peptide 146 of the other degron 142 of the targetprotein 140 is configured to be cleaved from the target protein 140 whenthe target protein's 140 cut site 125 specific for the second protease120 is cleaved by the second protease 120, wherein the target protein140 is configured to be destabilized by cleavage of its cut site 125specific for the second protease 120, wherein cleavage of the targetprotein's 140 cut site 125 specific for the second protease 120 uncoversthe target protein's 140 other degron 142 thereby destabilizing thetarget protein 140. In some embodiments, the second target protein's 240other degron 242 includes a masking peptide 246 of the other degron 142of the second target protein 240 connected to the second targetprotein's 240 other degron 242, wherein the masking peptide 246 of theother degron 142 of the second target protein 240 prevents the secondtarget protein's 240 other degron 242 from destabilizing the secondtarget protein 140 when the masking peptide 246 of the other degron 142of the second target protein 240 is present on the second target protein240, wherein the masking peptide 246 of the other degron 142 of thesecond target protein 240 is configured to be cleaved from the secondtarget protein 240 when the second target protein's 240 cut site 115specific for the first protease 110 is cleaved by the first protease110, wherein the second target protein 240 is configured to bedestabilized by cleavage of its cut site 115 specific for the firstprotease 110, wherein cleavage of the second target protein's 240 cutsite 115 specific for the first protease 110 uncovers the second targetprotein's 240 other degron 242 thereby destabilizing the second targetprotein 240.

In some embodiments, the synthetic protein circuit comprises an XNORlogic gate. In some embodiments, the synthetic protein circuit includesa third protease 130 including a cut site 115 specific for the firstprotease 110, a cut site 125 specific for the second protease 120, andone or more optional association domains 158, 159 of the third protease130, wherein the third protease 130 is configured to be deactivated bycleavage of either of its cut sites 115, 125; wherein the target protein140 further includes a second degron 142 of the target protein, a cutsite 115 specific for the first protease 110, a cut site 125 specificfor the second protease 120, and two cut sites 135, 135 specific for thethird protease 130, and wherein the target protein 140 is stabilized bycleavage of: its cut site 115 specific for the first protease 110 andits cut site 125 specific for the second protease 120, or both of itscut sites 135, 135 specific for the third protease 130. Othercombinations may also be included such as follows: 115 and the left 135,or 125 and the right 135.

In some embodiments of the synthetic protein circuit, the third protease130 further includes a first domain 138 of the third protease 130 and asecond domain 139 of the third protease 130; wherein the first domain138 of the third protease 130 includes the cut sites 115, 125 specificfor the first and second proteases 110, 120 and the optional associationdomain 158 of the third protease 130; wherein the second domain 139 ofthe third protease 130 includes another cut site 115 specific for thefirst protease 110, another cut site 125 specific for the secondprotease 120, and an optional complementary association domain 159 ofthe third protease 130; and wherein the third protease 130 is configuredto be deactivated by cleavage of any of its cut sites 115, 115, 125,125. In some embodiments, the target protein's 140 cut site 115 specificfor the first protease 110 and one of the target protein's 140 two cutsites 135, 135 specific for the third protease 130 separate the targetprotein's 140 first degron 141 from a part 144 of the target protein140; and wherein the target protein's 140 cut site 125 specific for thesecond protease 120 the other of the two cut sites 135 specific for thethird protease 130, and the association domain 159 of the target protein140 separate the target protein's 140 second degron 142 from the part144 of the target protein 140.

In some embodiments of the synthetic protein circuit, the system orsynthetic protein circuit comprises a bandpass circuit or filter, or anadaptive pulse circuit such as is shown, exemplified, or described inFIGS. 3A-3H. In some embodiments of the bandpass circuit or filter, asecond protease 120 is tuned by a third protease 130. In otherembodiments, a first protease is tuned by a second, third, or fourthprotease. In accordance with some embodiments, any protease may tuneanother protease. Some embodiments include a system such as a syntheticprotein circuit, including: a first protease 110; a second protease 120;and target proteins 140 each including: a first degron 141 of the targetprotein 140 that destabilizes the target protein 140 when present on thetarget protein 140 by enhancing degradation of the target protein 140, acut site 115 specific for the first protease 110 between the degron 141of the target protein 140 and a part 144 of the target protein 140,wherein the target protein 140 is configured to be stabilized bycleavage of its cut site 115 specific for the first protease 110, and acut site 125 specific for the second protease 120 connected to anotherdegron 142 of the target protein 140, wherein the target protein 140 isconfigured to be destabilized by cleavage of the cut site 125 specificfor the second protease 120 regardless of whether the first degron 141of the target protein 140 is present on the target protein 140. In someembodiments, the other degron 142 of each target protein 140 includes aconditional N-end degron.

Some embodiments include a third protease 130 including a cut site 125specific for the second protease 120, wherein the third protease 130 isconfigured to be deactivated by cleavage of its cut site 125 specificfor the second protease 120; and wherein the second protease 120includes a cut site 135 specific for the third protease 130, wherein thesecond protease 120 is configured to be deactivated by cleavage of itscut site 135 specific for the third protease 130.

In some embodiments of the synthetic protein circuit, the secondprotease 120 further includes a first domain 128 of the second protease120, a second domain 129 of the second protease 120, a firstcomplementary association domain 159, and an optional secondcomplementary association domain 159 c of the second protease 120connected to the first or second domain 128, 129 of the second protease120; wherein the first domain 128 of the second protease 120 includesthe cut site 135 specific for the third protease 130; wherein the seconddomain 129 of the second protease 120 includes another cut site 135specific for the third protease 130; wherein the first complementaryassociation domain 159 of the second protease 120 optionally includestwo parts 159 a, 159 b of the complementary association domain 159 ofthe second protease 120, each part 159 a, 159 b of the complementaryassociation domain 159 of the second protease 120 connecting to one ofthe second protease's 120 cut sites 135 specific for the third protease130; and wherein the second protease 120 is configured to be deactivatedby cleavage of either of its cut sites 135, 135.

In some embodiments of the synthetic protein circuit, the third protease130 further includes an optional association domain 159 of the thirdprotease 130, and wherein cleavage of the third protease's 130 cut site125 by the second protease 120 removes at least part of a cleavagedomain 139 of the third protease 130, thereby deactivating the thirdprotease 130.

In some embodiments of the synthetic protein circuit, the stability ofthe target proteins 140 includes an analog behavior that is dependent ona concentration of the first protease 110, wherein a higherconcentration of the first protease 110 has a greater stabilizing effecton the target proteins 140 than a lower concentration of the firstprotease 110. In some embodiments, the stability of the target proteins140 includes an analog behavior that is dependent on a concentration ofthe second protease 120, wherein a higher concentration of the secondprotease 120 has a greater destabilizing effect on the target proteins140 than a lower concentration of the second protease 120. In someembodiments, the concentration of the second protease 120 is decreasedby a higher concentration of the third protease 130 as compared to alower concentration of the third protease 130, or by a higher amount ofa nucleic acid encoding the third protease 130 as compared to a loweramount of a nucleic acid encoding the third protease 130. In someembodiments, the analog behavior of the target protein 140 that isdependent on a concentration of the second protease 120 is more sharpand/or includes a greater threshold for destabilizing the target protein140 at a higher concentration of the third protease 130 as compared to alower concentration of the third protease 130, or at a higher amount ofa nucleic acid encoding the third protease 130 as compared to a loweramount of a nucleic acid encoding the third protease 130.

In some embodiments of the synthetic protein circuit, the first protease110 further includes a first domain 118 of the first protease 110 and asecond domain 119 of the first protease 110; wherein the first domain118 of the first protease 110 connects to a first conditionaldimerization domain 368 of the first protease 110; wherein the seconddomain 119 of the first protease 110 connects to a second conditionaldimerization domain 369 of the first protease 110; wherein the first andsecond conditional dimerization domains 368, 369 of the first protease110 are configured to dimerize with each other upon binding a dimerizingagent 367. In some embodiments, the conditional dimerization domains368, 369 of the first protease 110 each include one of an FK506 bindingprotein (FKBP), GyrB, GAI, Snap-tag, eDHFR, BCL-xL, CalcineurinA (CNA),CyP-Fas, FRB domain of mTOR, GID1, HaloTag, TIR1, auxin inducibledegron, and/or Fab (AZ1). In some embodiments, the dimerizing agent 367includes FK1012, FK506, FKCsA, Rapamycin, Coumermycin, Gibberellin,HaXS, TMP-HTag, auxin, or ABT-737. In some embodiments, at least one ofthe conditional dimerization domains 368, 369 and/or the dimerizingagent 367 include a leucine zipper motif or a complementary leucinezipper motif, a scaffold protein or a fragment thereof, ascaffold-binding motif, an antibody, an epitope, tetratricopeptiderepeat, a tetracopeptide repeat-binding motif, a G-protein-coupledreceptor, a β-arrestin, and/or a G protein.

Some embodiments relate to a system such as a synthetic protein circuit,including: a first protease 110; a second protease 120; and a targetprotein 140 including: one or more cut sites specific for a first,second, and/or third protease, and a degron of the target protein 140configured to stabilize or destabilize the target protein 140 based onits configuration with one or more of the target protein's 140 cut sitesspecific for the first, second, and/or third proteases. In someembodiments, the first protease 110 further includes a first domain 118of the first protease 110 and a second domain 119 of the first protease110; wherein the first domain 118 of the first protease 110 connects toa first conditional dimerization domain 368 of the first protease 110;wherein the second domain 119 of the first protease 110 connects to asecond conditional dimerization domain 369 of the first protease 110;wherein the first and second conditional dimerization domains 368, 369of the first protease 110 are configured to dimerize with each otherupon binding a dimerizing agent 367.

In some embodiments of the system or synthetic protein circuit, theanalog behavior of the target protein 140 includes a bandpass behavior.

Some embodiments relate to a nucleic acid encoding all or a portion ofthe system or synthetic protein circuit described herein. In someembodiments, the nucleic acid includes DNA. In some embodiments, the DNAincludes a vector configured for transient expression in a cell. In someembodiments, the DNA includes an expression construct configured tointegrate into a host cell's DNA. In some embodiments, the nucleic acidincludes RNA such as an mRNA.

Methods

Some embodiments relate to a method, including: providing a reactionsolution with a protease or compound protease as described herein, andan enzyme such as a protease or compound protease or an enzyme describedherein; and subjecting the reaction solution to a condition that allowsthe enzyme to cleave the cut site 515 of the compound protease 520. Insome embodiments, providing the reaction solution comprises providing areaction solution in vitro. Some embodiments include providing thereaction solution to a cell or to cells.

Some embodiments relate to a method of activating a signaling pathway ina cell, including providing to the cell a synthetic protein circuit or anucleic acid encoding the synthetic protein circuit, the syntheticprotein circuit including: a protease 410 including a first part 418 ofthe protease 410 and a second part 419 of the protease 410, the firstpart 418 of the protease 410 connecting to a signaling protein 471, andthe second part 419 of the protease 410 connecting to a binding protein472 that binds to an activated form of the signaling protein 471,wherein the first part 418 and the second part 419 are configured toform an active protease 410 when the binding protein 472 binds to theactivated form of the signaling protein 471; and an effector protein 480including a cut site 415 specific for the protease 410, wherein theeffector protein 480 configured to be activated by cleavage of its cutsite 415 specific for the protease 410. An example of utilizing such amethod is shown in FIGS. 4A-4E. In some embodiments, the syntheticprotein circuit further includes a second protease 120 that inactivatesthe first protease 410 and/or the effector protein 480. In someembodiments, the signaling pathway includes a cell death pathway. Insome embodiments, the signaling protein 471 includes a signaltransduction protein such as Ras or a fragment thereof. In someembodiments, the binding protein 472 includes Raf or a fragment thereofsuch as a Ras-binding domain (RBD). In some embodiments, the effectorprotein 480 includes a protease, a cell death protein such as a caspase,an immunomodulatory, or a specific antigen. In some embodiments, themethod includes the use of a mutual inhibition motif such as a bandpassfilter or adaptive pulse circuit as described herein.

Further Embodiments

As provided herein, synthetic protein-level circuits allows forengineering of powerful new cellular behaviors. Rational protein circuitdesign is facilitated by a composable protein-protein regulation system,in which individual protein components can regulate one another tocreate a variety of different circuit architectures. Here, it is shownthat engineered viral proteases can function as composable proteincomponents, which can together implement a broad variety ofcircuit-level functions in mammalian cells. In some versions of thissystem, termed CHOMP (Circuits of Hacked Orthogonal Modular Proteases),input proteases dock with and cleave target proteases to inhibit theirfunction. These components can be connected to generate regulatorycascades, binary logic gates, and dynamic analog signal-processingfunctions. To demonstrate the utility of this system, a circuit wasrationally designed that induces cell death in response to upstreamactivators of the Ras oncogene. Because CHOMP circuits can performcomplex functions yet be encoded as single transcripts and deliveredwithout genomic integration, they offer a scalable platform tofacilitate protein circuit engineering for biotechnologicalapplications. According to some embodiments, these engineered proteasesenable programmable protein-level circuits that implement diversefunctions in mammalian cells.

Synthetic biology allows for rational design of circuits that confer newfunctions in living cells. Many natural cellular functions areimplemented by protein-level circuits, in which proteins specificallymodify each other's activity, localization, or stability. For example,caspase-mediated programmed cell death is regulated by a circuit ofproteases that activate one another through cleavage. Synthetic proteincircuits could provide advantages over gene regulation circuits,including faster operation, direct coupling to endogenous pathways,single transcript delivery, and function without genomic integration(FIG. 1D).

A challenge is designing ‘composable’ protein components whose inputsand outputs are of the same type, so that they can form a wide varietyof protein circuits, much as a few electronic components can be wired toproduce a variety of electronic circuits (FIG. 1D). While naturalprotein domains have been combined to generate proteins with hybridfunctions or to re-wire cellular pathways for research and biomedicalapplications, the lack of composability has limited the ability todesign protein-level function in living cells.

Viral proteases are useful for such systems. Many of them exhibit strongspecificity for short cognate target sites, which can be recognized andcleaved in various protein contexts. Natural viral diversity providesmultiple proteases with distinct specificities. Viral proteases can beused with degrons to control protein stability. They can also activatetranscription factors, synthetic intein zymogens, and other proteases ina purified protein system.

One protease used herein was the tobacco etch virus protease (TEVP). Toquantify TEVP activity, a reporter (target protein) was designed inwhich a cognate cleavage site (tevs) is inserted between a Citrinefluorescent protein and a dihydrofolate reductase (DHFR) degron, whichcan be inhibited by trimethoprim (TMP) as a positive control (FIG. 1E).Human embryonic kidney (HEK293) cells were transfected with plasmidsexpressing different combinations of TEVP, the reporter, and an mCherryco-transfection marker, and cells were analyzed by flow cytometry. ThemCherry signal was used to select highly transfected cells, which showedthe largest separation of basal reporter fluorescence from cellularautofluorescence to maximize the observable dynamic range of thereporter (FIG. 1E and FIG. 5A). Treating cells with TEVP stronglyincreased reporter abundance to levels similar to those obtained by TMPinhibition of the degron (FIG. 1E, FIG. 5B, left). A complementaryrepressible reporter (target protein) was also designed in which TEVPcleavage exposes a destabilizing N-terminal tyrosine residue (FIG. 1F,FIG. 5B, right). These designs generalized in a straightforward way tothe related tobacco vein mottling virus protease (TVMVP) and with somemodifications to the unrelated hepatitis C virus protease (HCVP) (FIG.5C, 1G). Furthermore, measuring activation of each reporter in responseto each protease revealed limited cross-activation (FIG. 1G). Thus,according to some embodiments, at least three viral proteases can beused to orthogonally increase or decrease cognate reporters.

Protease-protease regulation was achieved to enable design of complexcircuits. The degron strategy used for the reporters failed to producestrong regulation, possibly because proteases may cleave degrons withinthe same protease molecule with relaxed specificity. Instead, a schemewas designed that regulates protease activity, rather than abundance.Antiparallel hetero-dimerizing leucine zipper domains were incorporatedto each half of a split TEVP to reconstitute its activity (FIG. 1H,left). The inventors also inserted HCVP cleavage sites between theleucine zippers and TEVP, to allow HCVP to inhibit TEVP. Finally, theinventors fused a leucine zipper (complementary to one of the zippers onsplit TEVP) to HCVP, thus enhancing its ability to dock with, andinhibit its TEVP target (FIG. 5E, left). This design successfullyproduced repression of TEVP by HCVP (FIG. 1H, left).

To generalize this design, a similar TEVP variant repressed by TVMVP wasengineered (FIG. 5E, right). Based on its sequence similarity to TEVP(FIG. 5F), TVMVP variants repressed by either HCVP (FIG. 1H, right) orTEVP (FIG. 5G) were also engineered. To make these designs more compact,the inventors linked the two halves of each regulated protease with asingle leucine zipper flanked by cleavage sites for the input protease,creating single-chain repressible proteases (FIG. 1I and FIGS. 5H, 5I).Similar approaches enabled engineering protease regulation of theunrelated protease HCVP using a different split strategy describedherein. In these constructs, cleavage by either TEVP or TVMVP stronglyreduced HCVP activity, enabling signal propagation through three-stageprotease cascades (FIG. 1J and FIG. 5J). Together, this strategyestablished a composable protease regulation system.

Using this system, core circuit functions were designed, starting withBoolean logic. The inventors identified three design principles thattogether would be sufficient to enable all 8 two-input gates: First,incorporation of a consecutive pair of distinct cleavage sites between adegron and a target protein can implement OR logic, since cleavage ofeither site is sufficient to stabilize the protein (FIGS. 2A, 2B and6A). Second, to implement AND logic, the inventors flanked the targetprotein with FKBP and DHFR degrons on the N- and C-termini,respectively, each removable with a distinct cleavage site. On theN-terminus, a leucine zipper facilitated input protease docking. In thisdesign, removal of both degrons stabilized the protein (FIGS. 2A, 2C and6A). Third, to implement negation, the inventors either used the N-enddegron strategy (FIG. 1F) or propagated signals through an intermediateprotease repression step (FIG. 1H). Co-transfection of each basic gate(OR, AND, and NOR as a specific case of negation) with varyingconcentrations of its inputs revealed the expected logic functions(FIGS. 2A, 6B). Further, varying the concentration of the reporterplasmid enabled tuning of output levels without disrupting the logicalcomputation, facilitating matching of input and output levels in morecomplex circuits (FIG. 6C). Finally, by utilizing the HCVP inhibitorasunaprevir (ASV) and a rapamycin-induced TEVP, the inventors found thatthese gates could also be controlled by small molecule inputs (FIG. 7A).These results thus show that three core gates exhibit robust and tunableoperation across multiple input methods.

Next, these combined principles to were used to design and validate theother two-input gates (FIGS. 2A and 6A). Furthermore, to test whetheroutput from one gate could be directly used as input to a subsequentgate, the inventors constructed a more complex nested NOR function usingadditional orthogonal proteases from soybean mosaic virus (SMVP) andherpes simplex virus (HSVP) (FIG. 7B). The output from this system wasconsistent with that expected from the logical function NOR(TEVP,NOR(SMVP, HSVP)) (FIG. 7B).

Beyond Boolean logic, analog signal filtering can allow for manycellular functions, such as the ability to selectively respond tospecific input concentration ranges. The incoherent feed-forward loop(IFFL) motif, in which an input both activates and inhibits the sametarget, provides a simple implementation for this function. Inspired bythe IFFL, the inventors combined an activating arm, in which TEVPremoves a C-terminal degron, with a repressing arm, in which TVMVPreveals a destabilizing N-end tyrosine (FIG. 3A). To tune the positionand sharpness of the bandpass, the inventors also introduced a positivefeedback loop based on reciprocal inhibition between HCVP and TVMVP onthe repression arm, such that the amount of HCVP expression sets athreshold for TVMVP activity (FIG. 3A).

To characterize this bandpass circuit, the abundance of TEVP and TVMVPwere considered as input, and varied it through the concentration oftransfected DNA, which correlated linearly with protein abundance (FIG.8A). The individual activating and repressing arms of the circuitgenerated increasing and decreasing responses, respectively, toincreasing amounts of TEVP and TVMVP (FIG. 3B, 3C). Addition of HCVPincreased both the threshold and the sharpness of the response to TVMVPtitration (FIG. 3C). Combining the two arms into a single circuitgenerated the anticipated bandpass behavior, when the inventorsco-varied TEVP and TVMVP expression through either different amounts ofplasmid (FIG. 3D) or 4-epitetracycline (4-epi Tc) induction (FIG. 8B).Finally, varying the abundance of HCVP tuned the position and amplitudeof the bandpass response (FIG. 3D and FIG. 8B). These resultsdemonstrate rational engineering of tunable analog bandpass filters.

Temporal signal processing, such as adaptation to a change in input,plays a role in some biological systems. To engineer adaptation withCHOMP, the inventors designed an IFFL, containing the 3-step cascade(FIG. 1J) to introduce a delay in the repressing arm relative to that ofthe activating arm (FIG. 3E). To enable sudden induction, the inventorsadopted the rapamycin-induced TEVP used for the logic gates (FIGS. 7A,8C). To facilitate dynamic readout of circuit output in individual cellsthe inventors used a far-red fluorescent protein (IFP) that issynthesized in a non-fluorescent state, but can be post-translationallyswitched on by TEVP (FIG. 8D, left). The inventors also added aconditional N-end degron to enable repression by TVMVP (FIG. 3E).

The inventors encoded the entire pulse-generation circuit as a singleopen reading frame, with interleaved 2A “self-cleaving” peptides toseparate distinct protein components (FIG. 3F). This gene was thenstably incorporated in the genome. The inventors used flow cytometry toanalyze the response of the reporter in a single clone over time afterrapamycin addition. Cells exhibited the expected adaptive dynamics, witha rise in fluorescence on a timescale of hours and a subsequent decay tobaseline over ˜1 day (FIG. 8D, right). To obtain a direct view ofdynamics in individual cells, the inventors also analyzed the same cellline by time-lapse fluorescence microscopy (FIG. 3G). Analysis ofindividual cells revealed similar adaptive dynamics, respondingmaximally at 269±68 (mean±s.d.) min after rapamycin addition, decayingto 50% of their peak values over the subsequent 491±170 min, andeventually reaching fluorescence similar to that before induction (FIG.3H). These results demonstrate the design of single-gene multi-componentcircuits that generate dynamic signal responses.

By coupling directly to endogenous cellular outputs and inputs,protein-level circuits could act as programmable therapeutic devices. Asa proof of principle for such a strategy, the inventors designed acircuit to selectively kill cells with elevated activation of Ras, aprotein whose activity is increased in many cancers. More specifically,the inventors designed a core circuit that responds to upstreamactivators of Ras, such as SOS and EGFR, by activating an engineered TEVprotease, which in turn activates Caspase-3 (Casp3) to induce cell death(FIG. 4A, core circuit). The inventors then improved this circuit byincorporating additional proteases and interactions (FIG. 4A, fullcircuit).

To enable efficient protease-dependent induction of cell death at theplasma membrane, where Ras activation occurs, the inventors membranelocalized a TEVP-activated Casp3 variant by incorporating the 20 aminoacid membrane-targeting sequence) (‘mts’) from the C-terminus of humanH-Ras (FIG. 4A, box 2). Using flow cytometry, the inventors quantifiedthe effect of this Casp3 variant on cell numbers in terms of a‘reduction index’ whose value measures the relative reduction in cellnumber compared to a control condition (FIG. 9B). The membrane-targetedCasp3 decreased cell numbers when co-transfected with a similarlymembrane-localized TEVP variant (FIG. 4B), with higher efficiency thanthe original cytoplasmic Casp3 variant (FIG. 9D). Further, to allowbidirectional regulation by TEVP and TVMVP, the inventors alsoincorporated a TVMVP cleavage site adjacent to the mts tag (FIG. 4A, Box3), enabling membrane-localized TVMVP to remove Casp3 from the membraneand thereby attenuate its activation by TEVP (FIG. 4B).

Next, to couple Ras-activating inputs to TEVP, the inventors fused theN-terminal half of TEVP to Ras and its C-terminal half to theRas-binding domain (RBD) of Raf, which binds to the active form of Ras.In this design, upstream activators of Ras should reconstitute RasTEVP(FIG. 4A, core circuit and Box 1, FIG. 9C) and thereby activate Casp3.To validate this design, the inventors constructed a HEK293 cell linestably expressing a constitutively active Son of Sevenless (SOS_(CA))variant with a membrane-localization myristoylation signal and noinhibitory C-terminal region. Transfection of the core circuit reducedcell numbers both in this SOS_(CA) cell line and its parental controlline lacking ectopic SOS_(CA), but preferentially affected the SOS_(CA)cells (FIG. 4C, core circuit, and FIG. 9F). This selectivity requiredthe regulated Ras-RBD interaction (FIG. 9E). However, while this corecircuit provided some selectivity, it also exhibited a relatively highbackground rate of Casp3 activation in the control cells.

To improve the circuit's selectivity, the inventors incorporated aTVMVP-TEVP reciprocal inhibition motif (FIG. 9A, boxes 4 and 5) similarto the one used in the bandpass circuit, as well as feed-forwardrepression of Casp3 activation by TVMVP (FIG. 4A, Box 3). In this “fullcircuit” design, TVMVP should suppress activation of Casp3 in controlcells, both directly and indirectly through TEVP. By contrast, inSOS_(CA) cells, elevated activation of TEVP should override theinhibitory effects of TVMVP. The full circuit indeed improvedselectivity (FIGS. 4C, 9G, 10A). More specifically, expressing TVMVP inamounts comparable to, but lower than those of TEVP nearly abolishedoff-target effects in control cells, while retaining most of theon-target reduction in cell number (FIGS. 4C, 9G).

Further, the inventors encoded the full 4-protein circuit on a singletranscript, optimizing the relative abundance of components withinternal ribosome entry site (IRES) variants (FIGS. 4D, 10B), andtransfected it into a mixed population of SOS_(CA) and control cells. Atits optimal concentration (FIG. 10C), the single-transcript circuitreduced the number of SOS_(CA) cells by ˜40%, approaching the ˜50% upperlimit achieved by a positive control circuit that constitutivelyactivates Casp3 (FIG. 4D, and FIG. 4E, right). (The upper limit isconstrained by gene delivery and expression efficiency.) Importantly, itexhibited minimal effects on the control population (FIG. 4E, right).SOS_(CA)-dependent killing could also be observed using Annexin-Vstaining as an independent readout of apoptosis (FIG. 10D). Finally, totest the generality of the circuit, the inventors considered EGFRvIII,an oncogenic EGFR mutant found in glioblastoma and other cancer types.The single-transcript full circuit also selectively killed EGFRvIIIcells (FIG. 4E, left, and FIG. 10D). Together, these results show that aCHOMP circuit can be engineered to detect and kill in response toupstream activators of Ras through rational iterative designoptimization. Thus, according to some embodiments, the methods describedherein may be used in a biomedically relevant environment.

The results demonstrate how a set of composable protein regulators andcircuit design principles enable a remarkably broad range ofprotein-based circuits and functions. The use of a small number ofcomposable components shifts the design problem, in part, from the levelof the individual protein to the level of the protein circuit. In someembodiments where the operation of CHOMP components does not depend onhow they are expressed, they can be optimized through transienttransfections, accelerating the overall design-build-test cycle.

Some embodiments include protease-activating proteases, which in somecases simplify circuit designs and facilitate signal amplification. Someembodiments include multiple CHOMP inputs and outputs, and/or use directsensing of the activities of Ras and/or other oncogenes, and/or usecombinatorial sensing of multiple inputs.

Proteases can respond rapidly to an increase in input protease activity(FIG. 11). CHOMP circuits can also operate in parallel at specificsubcellular sites within a cell. In some embodiments where CHOMPcircuits have a relatively compact genetic design and do not useregulatory interactions with DNA, they are introduced intodifferentiated and/or post-mitotic cells with gene therapy vectorsand/or viruses, and/or improve the specificity of oncolytic virotherapy.Synthetically, hybrid circuits combining transcriptional ortranslational regulation with engineered proteases offers theprogrammability of base-pairing interactions together with protein leveloperation. For example, existing cancer-detection circuits mayconditionally express CHOMP components to increase specificity andcouple to protein-mediated inputs and outputs. In some embodiments,integrating these capabilities is used to, for example, produce smarttherapeutics or sentinels based on CHOMP circuits.

Where applicable, the experiments described in this section wereperformed in accordance with the materials and methods described inExample 1.

Characterization and Optimization of HCVP and its Reporter.

For HCV protease (HCVP), the inventors adopted a construct in which theprotease and its co-peptide are fused to create a more active singlechain protease. This HCVP initially showed more modest regulation thanthe other proteases, especially for the repressible reporter (FIG. 5D).The inventors reasoned that increasing the protease affinity to itstarget could improve its regulatory range. Indeed, incorporating a pairof hetero-dimerizing leucine zippers in the protease and its targetimproved regulation (FIG. 5D, right).

Characterization and Optimization of Circuits that Selectively ReduceRas-Activating Cells

To exclude the possibility that SOS_(CA) cells are generally moresensitive to Casp3 activation, the inventors first analyzedconstitutively dimerized split TEVP variants, one using leucine zippers,and the other adopting a RasG12V mutant that binds constitutively to RBD(FIG. 9E). When co-transfected with the TEVP-activatable Casp3, thesecontrol constructs displayed no selectivity for SOS_(CA) cells (FIG.9E), indicating that the regulated Ras-RBD interaction is necessary forthe selectivity observed in the main text (FIG. 4C).

To assess the contribution of each additional regulatory interaction inthe full circuit, the inventors systematically removed them one at atime, and compared their effects on control and SOS_(CA) cells to thefull circuit. Removal of Casp3 inhibition by TVMVP re-introducedsubstantial reduction in control cells (FIG. 10A, left), and removal ofTVMVP inhibition by RasTEVP increased survival in SOS_(CA) cells (FIG.10A, middle). By contrast, removal of RasTEVP inhibition by TVMVP had noeffect on survival in either control or SOS_(CA) cells (FIG. 10A,right). These results indicate that Arms 3,4 (FIGS. 9A, 10A) are majorcontributors to full circuit performance.

For single-transcript delivery of the full circuit, the inventorsinterposed a wild type internal ribosome entry site (IRES) between Casp3and RasTEVP coding sequences, followed by one of several IRES variantsequences (61) and then the TVMVP (FIG. 10B). Inspired by TVMVPtitration results (FIG. 9G), the inventors chose variants with ˜30% and˜70% of wild-type strength for the second IRES (55), and found that thecircuit functioned optimally with the ˜70% IRES (FIG. 10B).

Response of RasTEVP to EGF Stimulation

To assess the response of RasTEVP to a physiological ligand thatnormally activates the Ras pathway, the inventors stimulated cellsexpressing either RasTEVP or constitutively dimerized andmembrane-localized TEVP (negative control TEVP) with epidermal growthfactor (EGF). When co-transfected with a membrane-localized iTEVreporter, the control construct TEVP-mts exhibited minimal response toEGF stimulation, whereas RasTEVP displayed a modest response to EGF(FIG. 9C).

Comparison of Protease-Protease and Transcriptional Regulatory Dynamics

In the experiments described in this subsection, the inventors use aminimal model to address the question of how a simple transcriptionfactor regulatory step differs in dynamics from a simple proteaseregulatory step. To make a controlled comparison between the two kindsof regulation, the inventors assume that shared biochemical parameters,such as protein degradation rates, are similar in the two systems. Themain conclusion is that protease regulation can occur more rapidly thantranscriptional regulation but with timescales that depend on thedirection of regulation. By contrast, transcriptional regulation isexpected to be slower but show similar timescales in both directions ofregulation. While the inventors have considered typical biochemicalparameter values here, the inventors note that additional features ofany specific system, including feedback structure, could impact theirdynamic behavior. Additionally, the quantitative values of the resultingtimescales in general depend on the specific choice of biochemicalparameter values.

Protease-Protease Regulation.

The inventors modeled repression of one protease by another throughdirect cleavage, based on the scheme in FIG. 1E. The inventors assumethe concentration of the input protease, denoted P_(c), is maintained ata constant level, with its activity controlled by a small moleculeinput, as in the scheme of FIG. 3E. The output protease, denoted P, isproduced at a constant rate A, and undergoes first-order degradationwith rate γ_(p). The input protease cleaves the output protease at asingle cleavage site, converting it to a cleaved form, whoseconcentration is denoted P_(c), with a cleavage rate constant k. Thecleaved protease irreversibly dissociates at rate k_(d), and undergoesfirst-order degradation with rate γ_(p) for a total rate of eliminationof γ_(p)+k_(d). A single cleavage is assumed for simplicity, but thesame conclusions hold true for two independent cleavage sites, cleavageof either of which is sufficient to inactivate the output protease.

The reactions in the protease-protease model are as follows, wheredenotes ‘nothing’:

-   -   Synthesis of the output protease:

$\phi\overset{A}{\rightarrow}P$

-   -   Degradation of the output protease

$P\overset{\gamma_{p}}{\rightarrow}\phi$

-   -   Catalytic cleavage of the output protease:

${P_{0} + P}\overset{k}{\rightarrow}{P_{0} + P_{c}}$

-   -   Dissociation of the cleaved protease

$P_{c}\overset{k_{d}}{\rightarrow}\phi$

-   -   Degradation of the cleaved protease:

$P_{c}\overset{\gamma_{p}}{\rightarrow}\phi$

Assuming protease cleavage functions in a linear regime far fromsaturation, consistent with published K_(m) values and our bandpassmodeling, the reaction can be expressed as a set of ordinarydifferential equations (ODEs):

$\frac{dP}{dt} = {A - {{kP}_{0}P} - {\gamma_{p}P}}$$\frac{{dP}_{c}}{dt} = {{{kP}_{0}P} - {P_{c}\left( {k_{d} + \gamma_{p}} \right)}}$

Because the absolute value of the production rate A does not affect thedynamics of the system, the inventors arbitrarily set its value to 1Mh⁻¹. For the dissociation rate, the inventors assumed k_(d)=5 h⁻¹ basedon indirect measurements (71). For the protein degradation rate, theinventors assumed a biologically realistic value of γ_(p)=0.1 h⁻¹.

Based on our bandpass fits (FIGS. 3B-3D), cleavage by a protease, whenthe input protease activity is high, occurs at a rate comparable to therate of degron-mediated degradation (˜5 h⁻¹). The inventors also assumedthat the OFF input protease is 20-fold less active than the ON statebased on the dynamic range observed in FIGS. 1D-1J. (Note that the valueof this regulatory range does not affect our conclusions about thetimescales of regulation.) Finally, the inventors assumed thesmall-molecule-induced ON-OFF switch reaches steady-state much fasterthan the other reactions, so that the cleavage term can be approximatedby a step function, taking one of two possible values:

-   -   kP=0.25 h⁻¹ (input OFF) or 5 h⁻¹ (input ON)

To simulate output dynamics in response to changes in the input, theinventors first set the input protease to ON, and the output protease toits steady state value of P+P_(c). At t=10 h, the inventors switched theinput to OFF and simulated the equations for 70 h (10-80 h). Finally,the inventors switched the input back to ON and simulated another 70 h(80 h-150 h). FIG. 11 plots the resulting dynamics of the outputprotease, normalized to its maximum value. Note the asymmetric responsetime, which is faster for input OFF→ON switch than ON→OFF

$\left( {t_{\frac{1}{2}} = {0.32h^{- 1}\mspace{14mu}{{vs}.\mspace{11mu} 2.3}\mspace{11mu} h^{- 1}}} \right).$

Transcriptional Regulation.

As a comparison to protease regulation, the inventors modeled alogically equivalent transcriptional repression step. The inputtranscription factor was maintained at a constant concentration ofT_(G), with its activity assumed to be controlled by a small molecule,as with the protease. The input transcription factor regulates theoutput mRNA, T_(m), whose production follows a standard rate law:

$\frac{K}{K + T_{0}}{A_{m} \cdot T_{m}}$undergoes first-order degradation with rate γ_(m). The output proteinT_(p) is translated from the mRNA at rate A_(p), and degraded with rateγ_(p). The reactions are as follows:

-   -   mRNA synthesis:

$\phi\overset{\frac{K}{K + T_{0}}A_{m}}{\rightarrow}T_{m}$

-   -   mRNA degradation:

$T_{m}\overset{\gamma_{m}}{\rightarrow}\phi$

-   -   protein synthesis:

$T_{m}\overset{A_{p}}{\rightarrow}{T_{m} + T_{p}}$

-   -   protein degradation:

$T_{p}\overset{\gamma_{p}}{\rightarrow}\phi$

These reactions can be converted to ODEs for each of the components:

$\frac{{dT}_{m}}{dt} = {{\frac{K}{K + T_{0}}A_{m}} - {\gamma_{m}T_{m}}}$$\frac{{dT}_{p}}{dt} = {{A_{p}T_{m}} - {\gamma_{p}T_{p}}}$

Without loss of generality the inventors set the production rate A_(m)=1Mh⁻¹ and A_(p)=1 h⁻¹. The inventors used the same protein degradationrate as in the protease regulation case above: γ_(p)=0.1 h⁻¹. For mRNAdegradation, the inventors simulate two values at opposite extremes ofthe biological range for mammalian mRNA (72): γ_(m)=0.1 h⁻¹ (morestable), and 5 h⁻¹ (less stable). As above, the inventors also assumedthat the small-molecule-controlled input ON-OFF switch is much fasterthan the other reactions. To match the protease conditions, theinventors assumed T₀ also undergoes a 20-fold regulation, from T₀=0.5KOFF) to 10K (input ON), although the inventors note that the exactdynamic range of T₀ or the exact choice of the Hill function does notaffect output dynamics.

We simulated this simple model of transcriptional regulation with fastand slow mRNA degradation rates, following the same ON→OFF→ON inputtemporal profile used in the protease regulation case. To focus on thetimescale of regulation, the inventors normalized each curve to itsmaximal value. For transcriptional regulation,

$t_{\frac{1}{2}} = {7.2\mspace{14mu} h^{- 1}}$and 17 h⁻¹ for fast and slow mRNA decay, respectively, regardlesswhether the input undergoes ON→OFF or OFF→ON switch. When input switchesfrom ON to OFF, protease and transcriptional regulation occurs oncomparable timescales, although their difference is more apparent in theslower mRNA degradation case. When input switches from OFF to ON,however, protease regulation generates a much faster response timecompared to transcriptional regulation and the ON to OFF switch in theprotease regulation case (FIG. 11). Intuitively, the dynamics of eachprocess is limited by the slowest rate at which a species decays, whichis the relatively slow protein degradation rate for transcriptionalcontrol (or both protein and mRNA degradation rates when mRNA is morestable); in contrast, the output protease decays at a much faster ratebecause, in addition to regular protein degradation, it is also cleavedby input protease, and the rate is even higher when the input isswitched to its active state.

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Additional Embodiments

Programmable Protein Circuits in Living Cells

Synthetic biology approaches provide ways to program living cells toperform desired behaviors or functions. Synthetic biology could enable adiverse array of applications in biomedicine and biotechnology. Mostefforts so far have been based on genetic components that regulate eachother's transcription or translation. Synthetic circuits based onproteins could provide distinct capabilities, improving both circuitdelivery and function within the cell. The design and implementation ofprotein-level circuits has been hindered by the lack of a generalpurpose system in which proteins can be composed to regulate one anotherin a flexible, programmable manner.

Here we describe methods for engineering viral proteases to regulate oneanother and target proteins. We show that these methods enableengineering of circuits that perform regulatory cascades, binary logiccomputations, analog band-pass signal processing, generation of dynamicbehaviors such as pulsing, coupling to endogenous cellular states suchas oncogene activation, and the ability to control cellular behaviorssuch as apoptosis. The flexibility and scalability of this systemenables it to be reconfigured to implement a broad range of additionalfunctions. These circuits can also be encoded and delivered to cells inmultiple formats, including DNA, RNA, and at the protein level itself,enabling versatile applications without genomic integration ormutagenesis.

Applications could include the following:

-   -   Kill switches: The ability to kill engineered cells in response        to a signal is a key requirement for emerging cell based        therapies. Existing methods may produce toxicity or cell death        when not desired, i.e. in the absence of the kill switch input.        This invention includes the construction of kill switches in        which such effects can be suppressed through a protease-based        reciprocal inhibition motif or through feed-forward loop        structures.    -   Virally delivered synthetic circuits: The invention includes the        ability to deliver complex programmed protein-level functions        into cells using a variety of non-integrating vectors. This        capability avoids potential mutagenesis that occurs with gene        regulation based systems.    -   Oncolytic viral therapies: By encoding these protein level        circuits on oncolytic viruses, one could deliver functions that        specifically kill or inactivate tumor cells conditionally        depending on their state.    -   Gene drive payloads: Gene drive technology is a rapidly growing        area of biotechnology that enables the efficient super-Mendelian        propagation of genetic systems within mating populations of        organisms. Gene drive applications will depend on the ability to        package sophisticated functions in compact genetic systems. The        system described here can enable gene drives payloads that        perform such functions. For example, for insect vector control,        a protein-circuit that would specifically kill mosquitoes        infected by human pathogens such as Dengue virus.    -   Cell type specific control of cell fate. Regenerative medicine        requires precise manipulation of cell fate. These protein level        circuits can be transiently introduced into cells to control the        activation of fate regulating genes and thereby induce specific        cell fates. This activity can be coupled to modules within the        circuit that detect the state of the cell and make cell fate        control conditional on cell state. This avoids the problem of        activating the same genes in a heterogeneous cell population,        and also avoids permanent genetic modification.    -   Extracellular protein level feedback circuits that control blood        clotting. The system described here enables the design of        protein circuits that could function outside cells to detect        blood clots or other pathological conditions and trigger clot        removing functions.    -   Subcellular functions. The system described here enables the        design of protein circuits that function in specific subcellular        compartments or sites. These circuits could operate to modulate        the behavior of specific synapses or organelles such as        mitochondria.

The above are a few examples of the many possible applications of thissystem.

The ability to design synthetic circuits that can process signals andactuate cellular responses in a programmable manner could facilitateregenerative medicine, cell-based therapies, and other applications.Approaches based on transcriptional or translational regulation havemade remarkable advances towards this goal [refs]. However, generegulatory circuits can require potentially mutagenic genome integrationprocedures, are limited in operational speed and stability, and interactonly indirectly with key protein-level cellular activities. Syntheticprotein-level circuits based on a modular and composable set of proteincomponents could in principle circumvent these limitations. Here, weshow that viral proteases can be engineered to regulate one another in acomposable fashion, and then used to implement a broad variety ofcircuits including regulatory cascades, binary logic, analog signalprocessing, dynamic responses, and the sensing and conditional actuationof endogenous cellular pathways. The system, termed CHOMP (Circuits ofHacked Orthogonal Modular Proteases), combines protease-specificcleavage sites, cleavage-dependent degrons, split proteincomplementation, and modular dimerization domains. Multi-protein CHOMPcircuits can be encoded compactly as single transcripts and operatewithout genomic integration, avoiding the need for permanent geneticmodification and accelerating the design-build-test cycle. They thusoffer a flexible new platform for programming diverse protein-levelfunctionality in mammalian cells.

Many natural cellular functions are implemented by protein-levelcircuits, in which proteins specifically modify each other's activity,localization, or stability. For example, programmed cell death utilizesa circuit based on proteases (caspases) that activate one anotherthrough cleavage. The inherent modularity of some protein domainsenables their potential use as flexible components for synthetic circuitdesign. Varshaysky (1995) proposed a mechanism for a protein-level logicgate in which modular degrons regulate the stability of a protein inresponse to a combination of protein-level inputs. Modular proteininteraction domains have been used to re-wire endogenous proteincircuits, and couple their activities to non-natural inputs (I) [otherrefs]. Nevertheless, a general purpose system for protein-level circuitdesign has remained elusive due to the lack of a set of composableprotein components.

Viral proteases provide an ideal basis for protein circuit design (ref).They exhibit strong specificity for short cognate target sites, whichcan be recognized and cleaved in a variety of protein contexts (ref).Different proteases cleave with distinct site specificities, potentiallyenabling orthogonal regulation. Viral proteases can also be used inconjunction with degrons to control the stability of other proteins in amodular fashion (Voigt, others). Despite these natural advantages, ithas remained unclear whether viral proteases can be engineered toregulate one another to create more complex protein-level circuits (FIG.1L).

CHOMP Building Blocks

We started with tobacco etch virus (TEV) protease, which iswell-characterized and has been used in diverse biotechnologyapplications. To read out its activity, we constructed a reporter systemin which a Citrine fluorescent protein is fused to a DHFR degron thattargets the protein for degradation (2) (FIG. 1E). A TEV cleavage site(tevs) introduced between the degron and Citrine allows TEV protease toremove the degron, stabilizing the fluorescent protein. The DHFR degroncan also be stabilized by the drug trimethoprim (TMP), serving as apositive control. We transiently transfected HEK293 cells with plasmidsexpressing the protease, the reporter, and a constitutively expressedmCherry co-transfection marker, and analyzed cells by flow cytometry,gating on high levels of mCherry fluorescence (see Methods, FIG. 1E, andFIG. 5A). TEV protease strongly increased fluorescence of the reporterto levels comparable to those achieved by TMP (FIG. 1E). We also built acomplementary reporter inhibited by TEV protease. In this design, TEVcleavage reveals a destabilizing N-terminal tyrosine residue[Varshaysky, others] (FIG. 1F). TEV protease indeed reduced reporterfluorescence (FIG. 1F). Thus, TEV protease can be used to increase ordecrease the levels of another protein by removing or revealing degrons.

To enable the construction of multi-component circuits, we generalizedthe reporter designs to proteases from tobacco vein mottling virus(TVMV) (3) and Hepatitis C virus (HCV) (4) (FIGS. 5K, 5L). For HCV, weadopted a construct in which the HCV protease and its co-peptide arefused to create a more active single chain protease (5). This HCVprotease initially showed more modest regulation than the otherproteases, especially for the repressible reporter (FIG. 5L). Wereasoned that increasing the protease affinity to its target couldimprove its regulatory range. Incorporating a pair of hetero-dimerizingleucine zippers (6) in the protease and its target indeed improvedregulation by HCV protease (FIG. 5L). To test the orthogonality of theoverall set of proteases, we analyzed regulation of each activatablereporter by each protease (FIG. 1G). Together, these results establisheda set of positive and negative reporters for three orthogonal proteases,and introduced a mechanism for increasing protease target recognition.

Protease-protease regulation is essential for enabling the design ofcomplex circuits. We first incorporated the conditional degrons used inthe reporters within the proteases themselves. However, this strategyfailed to produce strong protease-dependent control of target proteaseactivity. Therefore, we took advantage of the previously describedability of dimerizing domains to reconstitute the activity of a splitTEV protease variant (7) (FIG. 1H). Attaching antiparallelhetero-dimerizing leucine zipper domains (6) to each half of the splitTEV protease reconstituted its activity, as expected (FIG. 1H). To allowHCV protease to inhibit the activity of reconstituted TEV protease, weinserted HCV cleavage sites between the leucine zippers and TEVprotease. Finally, inspired by the design of the repressible HCVreporter, we fused a complementary leucine zipper to the HCV protease,enhancing its ability to dock to, and regulate its TEV protease target(FIG. 5M [left]). This design successfully allowed inhibition of TEVprotease by HCV protease (FIG. 1J).

This design appeared to be general in terms of both input and output.Replacement of HCV cleavage sites with TVMV cleavage sites generated aTEV protease that could be inhibited by TVMV protease (FIG. 5M, right).Using a homology-guided approach to identify a corresponding split sitein the TVMV protease, we were able to similarly regulate TVMV proteaseby both HCV and TEV proteases (FIG. 1H and FIG. 5N). In a final designstep, to make the components as compact as possible, we engineered asingle chain implementation of this design. In this variant, we inserteda single leucine zipper flanked by cleavage sites for the input proteasebetween the two protease halves (FIG. 1I). This construct was also ableto transduce inputs from other proteases (FIG. 1I, 5O, 5P, 5R). AlthoughHCV protease lacks a known split site, we were able to effectively splitHCV protease by introducing the cleavage site between the major proteasedomain and the fused co-peptide and leucine zipper. In this construct,cleavage by an input protease strongly reduced HCV's ability to repressits target protease, enabling the design and construction of three-stepprotease cascades (FIG. 1J and FIG. 1Q)

To explore the capabilities of protease circuits, we next set out totest basic circuits for Boolean logic, analog filtering, and dynamicpulse generation. First, we first asked whether the engineered proteasescould be used to create binary logic gates, which are essential modulesin complex circuits. More specifically, we designed protease circuitsthat compute each of the eight non-trivial, two-input binary logicalfunctions. Remarkably, three design principles were sufficient to enableconstruction of all 8 logic gates: First, the incorporation of aconsecutive pair of distinct cleavage sites between a degron and thereporter can implement OR logic, since cleavage of either or both sitesis sufficient to eliminate a degron and thereby stabilize the protein(FIG. 2, OR). Second, flanking the reporter with degrons (FKBP[wandless] on the N-terminus, and DHFR on the C-terminus), eachremovable with a distinct cleavage site, implements AND logic, as bothdegrons must be removed to stabilize the protein (FIGS. 2A and 2B, OR).Third, as shown in FIGS. 1F and 1H, negation can be implemented eitherusing a cleavage site to reveal an N-end degron, or using anintermediate protease repressed by the input protease.

Using these principles, we designed and built each of the 8 possiblebinary logic gates (FIGS. 2A-2I and FIG. 6A). To characterize theirperformance in cells, we co-transfected each gate either with or withouteach of the two input proteases, HCV and TEV. Although they varied intheir quantitative performance, all of the gates showed the expectedqualitative behavior. For high-expressing cells, the best-performinglogic gates showed dynamic ranges (ratio of high to low output states)of 10-20-fold (OR, NOR, IMPLY). Other gates showed intermediate dynamicranges of ˜7-fold (AND, NAND, NIMPLY). The non-monotonic XOR and XNORshowed more modest dynamic ranges of 5.2 and 3.6, respectively.

The post-translational operation of the circuits could enable them tofunction not only when transiently transfected into cells, but also whenstably incorporated in the genome. We used the piggyBac transposonsystem to stably integrate the NOR gate, and verified that it behaves asexpected in response to combinations of transiently transfected inputprotease genes (FIG. 6D). Together, these results indicate that a smallnumber of design principles are sufficient to implement all binary logicoperations at the protein level, and suggest that the same circuit canfunction both as a stable integrant or as a transient transfection.

Beyond Boolean logic, many cellular behaviors require analog signalfiltering and, more specifically, the ability to selectively respond tospecific input concentration ranges [paradoxical, morphogen]. Theincoherent feed-forward loop (IFFL) motif, in which an input bothactivates and inhibits the same target, can perform bandpass filtering,but have not been implemented at the protein level [ref]. To constructan IFFL we combined an activating arm, in which TEV protease removes aC-terminal degron, with a repressing arm, in which TVMV protease revealsa destabilizing N-end tyrosine (FIG. 3A). In order to enable tuning, wealso introduced a double negative feedback loop between HCV and TVMVproteases on the repression arm such that the level of HCV expressionsets a threshold for TVMV activity (FIG. 3A). To characterize thesecircuits we varied the amount of transfected DNA expressing the inputTEV and TVMV proteases. This DNA concentration correlates with proteinexpression level (FIG. 8A).

The activating and repressing arms of the IFFL, taken individually,generated increasing and decreasing responses, respectively, toincreasing levels of TEV and TVMV protease (FIG. 3B, 3C). Addition ofHCV increased both the threshold and the sharpness of the response toTVMV protease concentration (FIG. 3C). Combining the two arms into asingle circuit generated the expected non-monotonic input-outputresponse (FIG. 3D and FIG. 8B). Finally, as predicted from a simplemodel of the circuit (FIG. 3D, Methods), varying the HCV expressionlevel tuned the width of the bandpass response (FIG. 3D and ExtendedData FIG. 8B). Together, these results demonstrate the ability torationally engineer tunable analog bandpass filters.

Temporal signal processing and, more specifically, adaptation to achange in input, play critical roles in diverse biological systems[refs]. To engineer adaptation, we designed a second IFFL circuit thatuses the 3-step cascade (FIG. 1J) to introduce a delay in the repressingarm relative to the activating arm (FIG. 3E and FIG. 3I). To analyzeresponses to rapid changes in inputs, we next constructed a split TEVprotease, in which addition of rapamycin reconstitutes TEV functionthrough attached heterodimerization domains (FIG. 8E) [ref]. Finally, toenable rapid read-out of cellular behavior we adopted a previouslyreported protease-triggered far red fluorescent protein, which issynthesized in a non-fluorescent state but can be switched on by TEVprotease [ref]. We encoded the entire circuit and reporter on a singlepolypeptide (FIG. 3F) and stably incorporated it in the genome(Methods).

Using flow cytometry, we analyzed the response of the reporter over timeafter rapamycin addition. We observed the predicted adaptive dynamics,with a rapid rise in fluorescence on a timescale of hours and asubsequent decay to baseline over a timescale of ˜1 day (FIGS. 3G and3H). To obtain a more direct view of the dynamics at the level ofindividual cells, we analyzed the same cell line using quantitativetime-lapse fluorescence microscopy (FIG. 3G, filmstrip, Movie S1).Consistent with flow cytometry, individual cells responded maximally att=210±XXX min after rapamycin addition, and then showed a monotonicdecrease in activity to near background levels (FIG. 3H, trace).Together, these results demonstrate the ability to program dynamicsignal responses.

Having established that CHOMP can enable programming of binary, analog,and dynamic protein-level behaviors, we next asked whether one couldrationally design CHOMP circuits that process endogenous inputs andcontrol endogenous outputs. As a design target, we focused on Ras, whichplays a strong role in diverse cancers and is difficult to target withconventional therapeutic strategies [refs]. More specifically, we soughtto use CHOMP to conditionally reduce cell survival depending on Rasactivation.

To couple proteases to the activation of cell death, we took advantageof a previously described cytoplasmic TEV-activated Caspase. Because Rasfunctions at the membrane, we first designed a membrane localizedTEV-activated Caspase 3 and a corresponding membrane-localized TEVp byfusing the C-terminal CAAX membrane localization peptide from Ras toboth TEV protease and Caspase 3 (FIG. 4F). This membrane-targetedCaspase 3 efficiently reduced cell numbers in a TEVp-dependent manner(FIG. 4G), outperforming the cytoplasmic variant (FIG. 10G). In order toadd negative regulation to Caspase 3, we further included a TVMVpcleavage site between Caspase 3 and the membrane localization tag. Withthis modification, membrane-localized TVMV protease expression couldrelease Caspase from the membrane and thus suppress its activation byTEV protease, providing dual modes of Caspase 3 regulation (FIG. 4G).Next, to couple Caspase 3 to an upstream input that activates Ras, wetook advantage of a Raf domain (RBD) that specifically binds to theactive form of Ras [ref]. We fused the N-terminal half of TEV proteaseto Ras and its C-terminal half to RBD so that Ras activation couldreconstitute TEV protease activity and thereby activate Caspase 3 (corecircuit, FIG. 4F). To validate this design, we stably expressed aconstitutively active SOS [ref] that activates Ras in HEK293 cells andcompared circuit behavior in this cell to the parental control linelacking ectopic SOS expression [ref]. Although the core circuitpreferentially reduced cell survival in SOS+ cells, we also observedsubstantial reduction in cell number in control cells (FIG. 4H, with 0ng TVMV).

To better discriminate between parental and SOS+ cells, we further addedTVMV protease, configuring it to inhibit both TEV protease and Caspase3, while also incorporating reciprocal inhibition of TVMV protease byTEV protease (FIG. 4F, full circuit). This addition was designed tosuppress basal activation of the Caspase 3 and sharpen the overallresponse to SOS. More specifically, at low levels of Ras activation (incontrol cells), TEV protease should reconstitute at low levels, leadingto correspondingly low levels of Caspase 3 activity, which are furthersuppressed by TVMV protease, both through its direct effect on Caspase 3and through its indirect effect on TEV protease. By contrast, atelevated levels of Ras activation (in SOS cells), TEV protease can bemore efficiently reconstituted, activating Caspase 3, while alsoinhibiting TVMV protease and thereby blocking its inhibitory effects.

To characterize this circuit, we co-transfected varying amounts of theTVMV protease together with the core circuit in SOS+ and control cells.In these experiments, TVMV protease improved discrimination of the SOSstate of cells in a dose-dependent fashion (FIG. 9I), with optimaldiscrimination occurring at TVMV plasmid concentrations of ˜33%-66% ofthe TEV plasmid concentration. Systematic removal of individualregulatory interactions revealed that TEV protease regulation of TVMVprotease and TVMV protease regulation of Caspase were major contributorsto improved selectivity (FIG. 10E). Together, these results demonstratethe design and implementation of a CHOMP circuit that can transduceendogenous inputs to physiological responses in a rational and tunablefashion.

A unique feature of the CHOMP framework is the ability to encode acomplete circuit on a single transcript, facilitating its delivery forpotential applications. In order to achieve single-transcript delivery,while preserving the ability to control the relative expression levelsof different components, we took advantage of IRES sequences of varyingstrengths [ref]. We encoded the full circuit as a single transcript witha wild-type IRES interposed between Caspase and TEV protease codingsequences, followed by one of several different variant IRES sequenceand then the TVMV protease (FIG. 10F). We found that the circuitfunctioned optimally when the second IRES strength was ˜70% of wild-type[ref]. We next transfected different concentrations of this optimalsingle-transcript circuit into a mixed population of SOS and controlcells. Despite the substantial variability inherent to transienttransfection, this single construct was able to preferentially reducethe SOS population (FIG. 4I and FIG. 10G). Delivered at optimalconcentration, the circuit had little effect on the parental cells, butreduced the SOS+ population by approximately 40% (FIG. 4I), showing howrational design of a relatively simple three-protease circuit can couplecellular responses to endogenous responses.

Discussion

Here we have engineered viral proteases to function as a set ofcomposable, post-translational regulatory components orthogonal toendogenous cellular pathways. These proteases can be designed toregulate one another to create protein-level “CHOMP” circuits thatimplement binary logic gates (FIGS. 2A-2I), analog signal processingfunctions (FIGS. 3A-3I), and dynamic responses (FIGS. 3A-3I). Byinterfacing proteases with endogenous cellular components, wedemonstrated, as a proof of principle, that a CHOMP circuit canselectively reduce the survival of cells overexpressing SOS, a key steptowards the long-standing challenge of targeting cells with elevated Rasactivation (FIGS. 4F-4I). [ref].

The CHOMP framework has several appealing features for general purposecellular computation. The circuits can be encoded in a compact manner,as a single transcript, without requiring transcriptional regulation ofindividual components. In the context of genomic integration, thisaspect avoids issues with transcriptional interference between circuitcomponents. Furthermore, CHOMP circuits can operate without genomicintegration, eliminating possible mutagenic consequences altogether andenabling an accelerated design-build-test cycle in which a circuit canbe constructed as one or more DNA molecules and immediately tested inliving cells. Our results also demonstrate unique functionalcapabilities of CHOMP circuits. By circumventing transcriptionalregulation, they respond faster than synthetic transcriptional circuitsin a “single-shot” response mode (FIGS. 3A-3I), although proteinreplenishment by translation can be rate-limiting for some dynamicoperations. They can also directly respond to, and regulate, endogenouspathway activities (FIGS. 4F-4I).

Additional features would further enhance the power and flexibility ofCHOMP. Protease-activating-proteases would simplify some circuit designsand facilitate signal amplification. Protein design strategies tocontrol the intrinsic nonlinearity (effective cooperativity) ofinput-output responses could enable the construction of interestingdynamical properties such as multistability [ref], oscillation [refs:E2000, Laurent], or excitability [refs]. Finally, all circuits shownhere were created with only 3 proteases, but additional orthogonalproteases would allow larger and more complex circuits[viral_protease_review].

We anticipate that the existing CHOMP framework will enable newcapabilities for synthetic biology applications. First, CHOMP circuitscan operate at the subcellular level, performing local computation atspecific sites within the cell. For example, by localizing components tosynaptic sites within the same neuron, one could engineer circuits thatmodulate individual synaptic strengths in response to synapticactivities. Second, CHOMP circuits have a relatively compact geneticdesign and do not require regulatory interactions at the DNA level.These properties could facilitate their introduction into differentiatedand even post-mitotic tissues and cells using gene therapy vectors orother viruses. In particular, they could improve the specificity ofoncolytic virus technology [ref]. Third, while we have focused onproteases here, CHOMP circuits are also compatible with other types ofsynthetic circuits. Hybrid circuits combining transcriptional ortranslational regulation with engineered proteases could offer theprogrammability of base-pairing interactions with the computationaladvantages of CHOMP. For example, existing cancer-detection circuits[refs] could conditionally express CHOMP components to increasespecificity and couple to protein-level inputs and outputs. In thefuture, one can envision CHOMP circuits acting as smart therapeutics[reviews] or sentinels [ref:collins], delivered by non-integratingviruses into cells, where they could be triggered by complexcombinations of cellular protein activities to enable sophisticatedcellular control.

Further Disclosure Relating to Some Figures.

FIGS. 1E-1L|Design Principles for CHOMP Components.

FIG. 1L, I have a dream. FIG. 1E, A protease-activatable reporter. Left,the reporter consists of a Citrine, a degron, and a TEV cleavage site inbetween. Citrine level is increased by TEV-mediated degron removal orTMP-mediated degron stabilization. Middle, distribution of Citrinesignal from the gated area in the scatter plot in FIG. 5A. Solid curvesindicate skew gaussian fits, and vertical dashed lines indicate peakpositions of the gaussians. The x coordinates of these dashed lines arereported as “fluorescent intensity”. Right, triplicate resultsquantified using the aforementioned procedure. FIG. 1F, Aprotease-repressible reporter. Citrine level is reduced bycleavage-mediated exposure of an N-end degron. FIG. 1G, Three orthogonalproteases. Fluorescent intensity in each square is normalized to theTMP-stabilized value of its corresponding reporter. FIG. 1H, Aprotease-repressible protease. TEV protease is split and thenreconstituted through dimerizing leucine zippers. Aleucine-zipper-tagged HCV protease docks to and cleaves the sitesinserted between TEV protease and leucine zippers, thus repressing TEV.Also FIG. 1H, The repressible design works for TVMV protease as well.FIG. 1I, A modified single-chain TEV protease still allows for dockingof and repressive cleavage by HCV protease. FIG. 1J, A three-proteaserepression cascade. Repressible HCV protease uses a different design,where TEV cleavage separates core HCV protease from its docking leucinezipper and activity-enhancing co-peptide. For FIGS. 1E-1L, 2A-2I, 5A,5K-5R, 6A, and 6D, The red lines indicate mean and the grey areasseparate the expected ON and OFF states.

FIGS. 2A-2I|Logic Gates.

TEV and HCV proteases serve as inputs, and Citrine as output. Thedesigns and performances for all eight non-trivial two-input logic gatesare listed. Fluorescent intensity in each panel is normalized to thecorresponding reporter stabilized with TMP (degron only at C terminus)or Shield+TMP (degrons at both termini).

FIGS. 3A-3I|Bandpass Filtering and Pulse Generation Using IncoherentFeed-Forward Loops.

FIG. 3A, Diagram of a bandpass circuit. TEV and TVMV proteaseexpressions are controlled through the amount of transfected DNA ordoxycycline-inducible enhancer. HCV protease is expressed at variousconstant levels to tune the threshold of the repression arm. FIG. 3B,Input-output curve of the activation arm. FIG. 3C, Input-output curve ofthe repression arm, in the presence of constant TEV and different HCVlevels. HCV protease increases the threshold and sharpens the curve.Curves in FIG. 3B and FIG. 3C are fitted to mechanistically generatedequations (see Methods). FIG. 3D, Bandpass behavior of the completecircuit. Increasing HCV protease shifts peak position and increases peakamplitude. Curves are predictions combining the fits from bothindividual arms. Data in FIGS. 3B-3D are normalized to TMP-stabilizedreporter. FIGS. 3E and 3I, Diagram of pulsing circuit. Rapamycin-induceddimerization of FKBP and FRB domains reconstitutes TEV protease.Cleavage of the reporter TEV site frees the tethered-away chromophoreand allows for IFP to mature. FIG. 3F, All pulse circuit componentsencoded on a single transcript. FIG. 3G, Filmstrips showing a singlecell stably expressing the pulse circuit, as well as Cerulean as asegmentation marker. After rapamycin induction, IFP signal (red)increases, followed by a slower decay, while Cerulean signal (blue)remains stable. FIG. 3H, Time traces of all observed single cells thatpass our analysis threshold (see Methods for details on normalizationand smoothing). Black line indicates medians at each time point.Rapamycin is added at 0 minute.

FIGS. 4F-4I|Selective Reduction of Ras-Activating Cells.

FIG. 4F, Layout of a circuit that selectively activates Caspase 3 inresponse to high Sos activity. The molecular mechanisms of eachregulatory edge are listed on the right. FIG. 4G, An engineered Caspase3 is activatable by TEV protease (diagram in FIG. 4F-2) and repressed byTVMV protease (diagram in 4F-5). We inferred the survival percentage ofcells that get the indicated components (see Methods). FIG. 4H, The corecircuit (4F-1, 4F-2) preferentially reduces survival in the presence ofectopic Sos, and adding TVMV-linked edges (4F-3, 4F-4, 4F-5) improvesselectivity. FIG. 4I, The entire circuit encoded on a single transcriptselectively reduces survival of Sos cells co-cultured with controlcells. Triplicate results from independent wells are displayed, and thebars indicate mean.

FIGS. 5A, 5K-5R|Characterization and Optimization of CHOMP Components.

FIG. 5A, Two representative scatter plots of flow cytometry data with(red) and without (blue) TEV. Citrine signal is represented on the yaxis and co-transfection marker Cherry on x. The dashed lines delineategating on high Cherry level that's analyzed in FIG. 1E. FIGS. 5K, 5L,Reporters activatable (left) and repressible (right) by TVMV (FIG. 5K)and HCV (FIG. 5L) proteases. The designs are the same as TEV reportersexcept for the specific cleavage sites. Repressible HCV reportercontains an extra leucine zipper, and exhibits stronger repression whenHCV protease is tagged with the complementary leucine zipper. FIG. 5M,Split TEV proteases repressible by HCV (left) and TVMV (right)proteases. Tagging the regulating proteases with a leucine zippergenerally enhances repression. FIG. 5N, Split TVMV protease repressibleby TEV protease. FIGS. 5O and 5R, Single-chain TEV protease repressibleby TVMV protease. FIG. 5P, Single-chain TVMV protease repressible by HCV(left) and TEV (right) proteases. FIG. 5Q, Three-protease cascade stillfunctions with an alternative layout.

FIGS. 6A, 6D|Logic Gates.

FIG. 6A, Diagrams of the molecular mechanisms for all gates. FIG. 6D,Performance of stably integrated NOR gate in two monoclonal cell lines.

FIGS. 8A, 8B and 8E|Characterization Related to Incoherent Feed-ForwardLoops.

FIG. 8A, Linear correlation between the amount of transfected DNA andCitrine expression from CMV promoter. FIG. 8b , Bandpass behavior inresponse to TEV and TVMV proteases expressed with different levels ofdox inducers. FIG. 8E, Pulsing behavior measured using flow cytometry(same stable cell line as in FIG. 3H). Each point represents thefluorescent signal in an individual well with indicated rapamycinexposure time. The value is extracted from the peak position ofskew-Gaussian-fitted histogram in far-red channel (same method as thepost-gating fit in the middle panel of FIG. 1E).

FIGS. 9D, 9H, 9I, and 10E-10G|Characterization and Optimization ofCircuits that Selectively Reduce Ras-Activating Cells.

a, Example. FIG. 9D, Cytoplasmic TEV-activatable Caspase 3 causeslimited reduction of cell survival in the presence of membrane-localizedsplit TEV protease reconstituted through leucine zippers (compare toFIG. 4G). FIG. 9H, The effects of TEV and Caspase doses on survivalreduction. FIG. 9I, Dose of TVMV protease tunes the circuit'sselectivity for Sos cells (first and fourth pairs of data points alsoshown in FIG. 4H). FIG. 10E, Testing the contribution of individualregulatory edges to selectivity. Left, removing TEV┤TVMV edge increasesoverall survival; middle, removing TVMV┤Casp3 edge reduces overallsurvival; right, removing TVMV┤TEV edge has no significant effect. FIG.10F, Using IRES mutants to tune TVMV expression level in a singletranscript. The mutant reported to express at ˜70% level of wild typeexhibits a balance between survival of control cells and reduction ofSos cells. FIG. 10G, Selective survival reduction in response todifferent doses of the optimal single-transcript circuit (with 70%IRES), delivered into co-cultured control and Sos cells (second pair ofdata points also shown in FIG. 4I).

REFERENCES

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EXAMPLES Example 1—Materials and Methods for Additional EmbodimentsRelating to Programmable Protein Circuits in Living Cells

Where applicable, the materials and methods described in this sectionwere used in any experiments described herein, unless otherwiseindicated herein. Some embodiments of the methods, compositions, andsystems described herein include materials and/or methods described inthis example.

Plasmid Construction

Constructs were generated using standard procedures. The backbones werelinearized using restriction digestion or PCR, and inserts weregenerated using PCR or gBlock synthesis (IDT). A list of plasmids usedis included in Table 1, and plasmids and maps are deposited withAddgene.

TABLE 1 Miscellaneous 0a PGK-H2BChe 0b CMV-TO-H2BCit 0cCMV-TO-Cer-HO1-FlpIn 0d CMV-TO-MSos-2A-H2BChe-FlpIn 0ePB-CMV-TO-EGFRvIII-IRES-nlsChe reporter 1a PB-PGK-Cit-tevs-DHFR 1bPB-PGK-teD-Cit 1c PB-PGK-Cit-tvmvs-DHFR 1d PB-PGK-tvD-Cit 1ePB-PGK-Cit-hcvs-DHFR 1f PB-PGK-ZhcD-Cit 1g PB-PGK-Cit-tehc-DHFR 1hPB-PGK-FKBP-Zte-Cit-hc-DHFR 1i PB-PGK-Cit-tvhc-DHFR 1jPB-PGK-FKBP-Zte-Cit-tv-DHFR 1k PB-PGK-teD-Cit-hcvs-DHFR 1lPB-PGK-ZhcD-Cit-tevs-DHFR 1m PB-PGK-FKBP-Ztetv-Cit-tvhc-DHFR 1nPB-PGK-tvD-Cit-tevs-DHFR 1o pcDNA3.1-tvD-Cit-tevs-DHFR 1pPB-CMV-TO-Casp3te 1q PB-CMV-TO-Casp3te-tv-cx 1r PB-CMV-TO-Casp3te-cx 1sPB-CMV-Cit-tehc-DHFR 1t PB-CMV-FKBP-Zte-Cit-hc-DHFR 1uPB-CMV-Cit-tvmvs-DHFR 1v PB-CMV-TO-lyn-tvDiTEV TEV protease 2aCMV-TO-TEVP 2b CMV-TO-nTEVPhcZ-2A-ZhccTEVP 2cCMV-TO-nTEVPtvhcZ-2A-ZhctvcTEVP 2d CMV-TO-TEVPZ 2eCMV-TO-nTEVPhcZhccTEVP 2f CMV-TO-nTEVPtvZtvcTEVP 2gCMV-TO-nTEVPtvR-2A-KtvcTEVP 2h CMV-TO-nTEVPZ-tv-cx 2i CMV-TO-ZcTEVP 2jCMV-TO-nTEVP-tv-ras 2k CMV-TO-Rbd-tv-cTEVP 2l CMV-TO-nTEVP-rasG12V 2mCMV-TO-nTEVP-ras 2n CMV-TO-Rbd-cTEVP 2o CMV-TO-rapTEVPZ 2pCMV-TO-RasTEVP TVMV protease 3a CMV-TO-TVMVP 3b CMV-TO-TVMVPZ 3cCMV-TO-nTVMVPhcZ-2A-ZhccTVMVP 3d CMV-TO-nTVMVPteZ-2A-ZtecTVMVP 3eCMV-TO-nTVMVPhcZhccTVMVP 3f CMV-TO-nTVMVPteZtecTVMVP 3gCMV-TO-nTVMVPtehcZ-2A-ZhctecTVMVP 3h CMV-TO-nTVMVPhcZhccTVMVPZ 3iCMV-TO-nTVMVPhcZhccTVMVPcx 3j CMV-TO-nTVMVPteZtecTVMVPcx HCV protease 4aCMV-TO-scHCVP 4b CMV-TO-ZscHCVP 4c CMV-TO-ZNS4AteHCVP 4dCMV-TO-ZNS4AtvHCVP 4e pcDNA3.1-ZNS4AtvHCVP 4f CMV-TO-ZNS4AhssbHCVPCombination 5a CMV-TO-ZscHCVP-TEVPZ 5bPB-CMV-TO-rapTEV-teHCV-hcTVMV-tvDiTEV-Neo 5cPB-CMV-TO-Casp-rastvTEV-teTVMV0 5d PB-CMV-TO-Casp-rastvTEV-teTVMV1 5ePB-CMV-TO-Casp-rastvTEV-teTVMV2 5f PB-CMV-TO-Casp-rastvTEVca-teTVMVd1Other proteases 6a CMV-TO-SMVPZ 6b CMV-TO-HSVPZ

Tissue Culture

The Flp-In™ T-REx™ 293 Cell Line (Human Embryonic Kidney cells thatcontain a single stably integrated FRT site at a transcriptionallyactive genomic locus, and stably expressing the tetracycline repressorprotein) was purchased from Thermo Fisher Scientific (R78007). Cellswere cultured in a humidity controlled chamber at 37° C. with 5% CO₂ inmedia containing DMEM supplemented with 10% FBS, 1 mM sodium pyruvate, 1unit/ml penicillin, 1 μg/ml streptomycin, 2 mM L-glutamine and 1×MEMnon-essential amino acids. 100 ng/mL doxycycline was added wheneverexpression is needed from a CMV-TO promoter. All stably integratedtransgenes were inducible with doxycycline, which was only added one daybefore characterization. Trimethoprim (TMP) was delivered at 1 μM.Rapamycin was delivered at 5 nM. Epidermal growth factor (EGF) wasdelivered at 25 ng/mL. SHIELD1 was delivered at 1 μM. ASV was deliveredat 3 μM. For bulk measurement of pulsing dynamics, cells were culturedin the presence of 40 μM biliverdin, and rapamycin was added atdifferent time points before preparation for flow cytometry. Forstimulation with EGF, cells were cultured to near 100% confluency beforetransfection, and, one day after transfection, exposed to 40 μMbiliverdin, 25 ng/mL EGF, and 100 ng/mL doxycycline for 6 hours prior toflow cytometry analysis.

Transient Transfection

293 cells were seeded at a density of 0.05×10⁶ cells per well of a24-well plate and cultured under standard conditions overnight. Thefollowing day, the cells were transfected with plasmid constructs usingLipofectamine 2000 (Thermo Fisher) as per manufacturer's protocol.

Flow Cytometry

Two days after transfection, cells were prepared for flow cytometry bytrypsinizing with 30 μL of 0.05% trypsin for 1 min at room temperature.Protease activity was neutralized by resuspending the cells in buffercontaining 70 μL of HBSS with 2.5 mg/ml Bovine Serum Albumin (BSA). Forcells stimulated with EGF, cells were resuspended in buffer containing70 μL of HBSS with 2.5 mg/mL BSA and 1 mM EDTA. Cells were then filteredthrough a 40 μm cell strainer and analyzed by flow cytometry (MACSQuantVYB, Miltenyi or CytoFLEX, Beckman Coulter). The inventors used theEasyFlow Matlab-based software package developed in-house by YaronAntebi to process flow cytometry data.

Annexin V Staining

Staining was performed using a standard kit (ThermoFisher A13201). Oneday after transfection, cell culture medium was removed from each well,and replaced with 7.5 μL FITC-conjugated annexin V within 150 μL bindingbuffer. After incubation in dark at 37° C. for 15 min, the stainingmedium was removed, and the cells trypsinized for flow cytometryanalysis.

Fluorescent Signal Quantification from Flow Cytometric Measurements

To maximize the observable reporter dynamic range, the inventorsselected and compared cells with the highest expression of theco-transfection marker, which showed the largest separation of basalreporter fluorescence from cellular autofluorescence. For each sample ina comparison group (experiments performed in the same batch and datashown on the same plot), the inventors calculated the 98 and 99.5percentiles of fluorescence of the co-transfection marker (mCherry inmost cases). The inventors identified the sample with the lowest 98percentile value, and used its 98 and 99.5 percentiles as lower andupper limits to gate on all samples. For all cells within the gate ineach sample, the inventors fit the distribution of the logarithm oftheir signal fluorescence (Citrine in most cases) with skew Gaussiandistributions, i.e. N*normcdf(x,m,k)*normpdf(x,m,s) in Matlab usingnon-linear least-square fitting, and reported the mode (peak position,representing the reporter level that's most likely to be observed) ofthe resulting fit (FIG. 5A). Here, the normcdf(x,μ,σ) and normpdf(x,μ,σ)functions are cumulative probability density and probability densityfunctions for a Gaussian distribution respectively, and the parameter nis a normalization factor, m=μ is the mean of the Gaussian function, s=σis the inverse standard deviation of the Gaussian, and k parameterizesskewness. No gating was performed on monoclonal cells with thegenomically integrated pulsing circuit, because, unlike transienttransfection, here expression variation is already limited.

Calculating Reduction Index from Flow Cytometric Measurements

To calculate the reduction of cell numbers, the inventors compared theeffects of various treatments on cell numbers, comparing eachmeasurement to a negative control transfected with only a fluorescentmarker, and using the size of the untransfected cell population forinternal normalization. To do this, the inventors proceeded in severalsteps: First, the inventors fit the distribution of the logarithm ofautofluorescence collected in the Citrine channel from mock transfectedcells with the MATLAB function N₀*normcdf(x,m₀,k₀)*normpdf(x,m₀,s₀)using non-linear least-square fitting. Here, the parameters no, m₀, s₀,and k₀ and functions normcdf( ) and normpdf( ) have the same meanings aselsewhere described herein. Reference values for m₀, s₀, k₀, were thusdetermined from measurement of autofluorescence in untransfected cellsand fixed for subsequent two-component model fits. Second, for eachtransfected well, the inventors fit the distribution of the logarithm ofCitrine signal with N₁*normcdf(x,m₀,k₀)*normpdf(x,m₀,s₀)+N₂*normpdf(x,m₂,s₂), where N₁, N₂, m₂, s₂ were free parametersand m₀, s₀, k₀ were fixed to values extracted from autofluorescence fit.The area under the curve N₁*normcdf(x,m₀,k₀)*normpdf(x,m₀,s₀) (“area a₀”and “area a” in FIG. 9B) corresponds to the number of untransfectedcells, which serves as an internal reference. Third, the inventorssubtracted the number of untransfected cells from the total number ofcells to get the number of transfected cells that survived (“area b₀”and “area b” in FIG. 9B). For each sample, the number of transfectedcells that survived was then normalized to the number of untransfectedcells, and the ratio between normalized survival number in thatcondition ((area a)/(area b) in FIG. 9B) and normalized survival numberin the Citrine-only control condition ((area a₀)/(area b₀) in FIG. 9B)was defined as survival percentage. Finally, the reduction index wasdefined as 1-survival percentage.

In experiments with SOS_(CA) cells, a small fraction of these cellssilenced their transgene expression during cell culture. To make surethat the inventors were only analyzing cells that do express a Rasactivator, the inventors gated on mCherry that's co-expressed withSOS_(CA), and excluded the mCherry-population. This co-expressed mCherrymarker was also utilized in co-culture experiments, to distinguishSOS_(CA)/EGFRvIII cells from control cells, so that the inventors couldcalculate their reduction index separately.

Mathematical Modeling of the Bandpass Circuit

To analyze the behavior of the bandpass circuit, the inventorsconstructed a minimal ordinary differential equation model representingthe key components and interactions within the circuit. The modelincorporated three types of interactions: protein production,first-order degradation, and cleavage by proteases. In the model,protease regulation of substrates is described by differential equationsof the following form:

$\begin{matrix}{\frac{d\lbrack{Substrate}\rbrack}{dt} = {A - {{k_{cat}^{Protease}\lbrack{Protease}\rbrack}\lbrack{Substrate}\rbrack} - {k_{dA}\lbrack{Substrate}\rbrack}}} & (1) \\{\frac{d\left\lbrack {Substrate}_{cleaved} \right\rbrack}{dt} = {{{k_{cat}^{Protease}\lbrack{Protease}\rbrack}\lbrack{Substrate}\rbrack} - {k_{dB}\left\lbrack {Substrate}_{cleaved} \right\rbrack}}} & (2)\end{matrix}$

Here, A represents the production rate of a proteolytic substrate,k_(cat) ^(Protease) represents the catalytic coefficient, assuming thatproteolysis can be described as a Michaelis-Menten reaction far fromsaturation, and the first-order degradation rates k_(dA) and k_(dB)represent degradation through basal cellular degradation pathways. Theserate constants can take higher or lower values depending on whether thesubstrate protein and its cleaved form are unstable or stable,respectively.

To simplify the analysis without loss of generality, the inventors setA=1 in the equations for fluorescent reporters, effectively usingarbitrary normalized units for the fluorescent protein concentrations.[Substrate] in the normalized version thus corresponds to [Substrate]/Ain the original version.

We first considered a Cit_(DHFR) reporter, whose DHFR degron can beremoved by TEVP with a coefficient k_(cat) ^(TE). In its initial form,the reporter degrades at rate k_(d1) (Equation 3), while its cleavedproduct, Cit, degrades at a rate k_(d2) (Equation 4).

$\begin{matrix}{\frac{{dCit}_{DHFR}}{dt} = {1 - {{k_{cat}^{TE}\lbrack{TEVP}\rbrack}\left\lbrack {Cit}_{DHFR} \right\rbrack} - {k_{d\; 1}\left\lbrack {Cit}_{DHFR} \right\rbrack}}} & (3) \\{\frac{dCit}{dt} = {{{k_{cat}^{TE}\lbrack{TEVP}\rbrack}\left\lbrack {Cit}_{DHFR} \right\rbrack} - {k_{d\; 2}\lbrack{Cit}\rbrack}}} & (4)\end{matrix}$

The steady-state solutions for Eqs. 3, 4 are:

$\begin{matrix}{{Cit}_{DHFR} = \frac{1}{{k_{cat}^{TE}\lbrack{TEVP}\rbrack} + k_{d\; 1}}} & (5) \\{{Cit} = \frac{k_{cat}^{TE}\lbrack{TEVP}\rbrack}{k_{d\; 2}\left( {{k_{cat}^{TE}\lbrack{TEVP}\rbrack} + k_{d\; 1}} \right)}} & (6)\end{matrix}$

Experimentally measured reporter fluorescence corresponds to the sumCit_(DHFR)=Cit. The absolute value of the independent variable [TEVP] isnot known. However, based on experiments in which protein expressionlevels correlated linearly with the amount of transfected plasmid (FIG.8A), the inventors substituted the concentration of transfected plasmid,p_(TE), for [TEVP] in all equations, effectively absorbing the constantof proportionality relating [TEVP] and p_(TE) into the k_(cat) ^(TE)values. With these simplifications, measured fluorescence can bewritten:

$\begin{matrix}{{Cit}_{total} = {{{Cit}_{DHFR} + {Cit}} = \frac{\frac{k_{cat}^{TE}p_{TE}}{k_{d\; 2}} + 1}{{k_{cat}^{TE}p_{TE}} + k_{d\; 1}}}} & (7)\end{matrix}$

Using Matlab's curve fitting toolbox, the inventors determined best fitvalues of the parameters k_(cat) ^(TE), k_(d1) and k_(d2) by fitting Eq.7 to the experimentally measured p_(TE)−Cit_(total) curve (FIG. 3B).

To model the repression arm of the bandpass circuit, the inventors musttake into account the mutual inhibitory activities of TVMVP and HCVP inthe circuit. These protease-protease equations take on the general formoutlined in Eqs 1, 2. However, because reporter and proteaseconcentrations are measured in different units (fluorescence and plasmidconcentration, respectively), their production rates cannot both bearbitrarily set to 1. Instead, the inventors denoted the proteaseproduction rate B, to account for the different units used for these twospecies. Specifically, for 1 unit of plasmid input to produce 1 unit ofprotease at steady-state, B must equal the degradation rate of theprotease multiplied by the amount of plasmid input (p_(Protease)), asshown below in Equations 8 and 9.

$\begin{matrix}{\frac{d\lbrack{TVMVP}\rbrack}{dt} = {{k_{dTV}p_{TV}} - {{k_{cat}^{HC}\lbrack{HCVP}\rbrack}\lbrack{TVMVP}\rbrack} - {k_{dTV}\lbrack{TVMVP}\rbrack}}} & (8) \\{\frac{d\lbrack{HCVP}\rbrack}{dt} = {{k_{dHC}p_{HC}} - {{k_{cat}^{TV}\lbrack{TVMVP}\rbrack}\lbrack{HCVP}\rbrack} - {k_{dHC}\lbrack{HCVP}\rbrack}}} & (9)\end{matrix}$

At steady-state, the concentration of TVMV protease can be expressed asa function of the plasmid inputs of TVMVP and HCVP:

$\begin{matrix}{\lbrack{TVMVP}\rbrack = \frac{W + \left( {W^{2} + {4k_{cat}^{TV}k_{dTV}^{2}k_{dHC}p_{TV}}} \right)^{\frac{1}{2}}}{2k_{cat}^{TV}k_{dTV}}} & (10)\end{matrix}$

where W≡k_(dTV)k_(cat) ^(TV)p_(TV)−k_(dHC)k_(dTV)−k_(cat)^(HC)k_(dHC)p_(HC). The reporter repressed by TVMVP is denoted Cit whennot cleaved (first-order degradation rate k_(d3)), and Cit_(Ndeg) whencleaved by TVMVP to expose an N-end degron (first-order degradation ratek_(d4)). We then used a procedure similar to Eqs. 3-7 to expressreporter expressions in terms of [TVMVP]:

$\begin{matrix}{{Cit} = \frac{1}{{k_{cat}^{TV}\lbrack{TVMVP}\rbrack} + k_{d\; 3}}} & \left( 11^{*} \right) \\{{Cit}_{Ndeg} = \frac{k_{cat}^{TV}\lbrack{TVMVP}\rbrack}{k_{d\; 4}\left( {{k_{cat}^{TV}\lbrack{TVMVP}\rbrack} + k_{d\; 3}} \right)}} & \left( 12^{*} \right) \\{{Cit}_{total} = \frac{\frac{k_{cat}^{TV}\lbrack{TVMVP}\rbrack}{k_{d\; 4}} + 1}{{k_{cat}^{TV}\lbrack{TVMVP}\rbrack} + k_{d\; 3}}} & \left( 13^{*} \right)\end{matrix}$

For all equations denoted with “*”, [TVMVP] takes the value defined inEq. 10.

We estimated the values of parameters, k_(cat) ^(HC), k_(cat) ^(TV),k_(dHC), k_(dTV), k_(d3), k_(d4), by fitting Eq. 13 to experimentallymeasured Cit_(total), p_(TV), and p_(Hc). (FIG. 3C).

To characterize the cooperativity caused by TVMVP-HCVP mutualinhibition, the inventors fit the repression curves in FIG. 3C with asigmoidal function:

$\begin{matrix}{{Cit}_{total} = \frac{C}{1 + \left( \frac{p_{TV}}{K} \right)^{n}}} & (14)\end{matrix}$The 95% confidence intervals for the Hill coefficient, n, were0.95±0.13, 2.0±0.4, and 2.4±0.5, for p_(HC) values of 0, 50, and 200 ng,respectively.

Finally, for the reporter that's simultaneously regulated by theactivation and repression arms, depending on whether the DHFR degron isremoved and whether the N-end degron is exposed, there are four possiblespecies Cit_(DHFR), Cit_(DHFR→Ndeg), Cit, and Cit_(Ndeg), thefirst-order degradation rates of which are denoted as k_(dA), k_(dB),k_(dC), and k_(dD), respectively. Similarly, the dynamics of these fourspecies can be expressed as:

$\begin{matrix}{\frac{{dCit}_{DHFR}}{dt} = {1 - {{k_{cat}^{TE}\lbrack{TEVP}\rbrack}\left\lbrack {Cit}_{DHFR} \right\rbrack} - {{k_{cat}^{TV}\lbrack{TVMVP}\rbrack}\left\lbrack {Cit}_{DHFR} \right\rbrack} - {k_{dA}\left\lbrack {Cit}_{DHFR} \right\rbrack}}} & \left( 15^{*} \right) \\{\frac{{dCit}_{{DHFR} + {Ndeg}}}{dt} = {{{k_{cat}^{TV}\lbrack{TVMVP}\rbrack}\left\lbrack {Cit}_{DHFR} \right\rbrack} - {{k_{cat}^{TE}\lbrack{TEVP}\rbrack}\left\lbrack {Cit}_{{DHFR} + {Ndeg}} \right\rbrack} - {k_{d\; B}\left\lbrack {Cit}_{{DHFR} + {Ndeg}} \right\rbrack}}} & \left( 16^{*} \right) \\{\frac{dCit}{dt} = {{{k_{cat}^{TE}\lbrack{TEVP}\rbrack}\left\lbrack {Cit}_{DHFR} \right\rbrack} - {{k_{cat}^{TV}\lbrack{TVMVP}\rbrack}\lbrack{Cit}\rbrack} - {k_{dC}\lbrack{Cit}\rbrack}}} & \left( 17^{*} \right) \\{\frac{{dCit}_{Ndeg}}{dt} = {{{k_{cat}^{TE}\lbrack{TEVP}\rbrack}\left\lbrack {Cit}_{{DHFR} + {Ndeg}} \right\rbrack} + {{k_{cat}^{TV}\lbrack{TVMVP}\rbrack}\lbrack{Cit}\rbrack} - {k_{dD}\left\lbrack {Cit}_{Ndeg} \right\rbrack}}} & \left( 18^{*} \right)\end{matrix}$

We summed the steady-state solutions of all species from these equationsto derive the final input-output equation for the bandpass circuit:

$\begin{matrix}{{{Cit}_{total} = \frac{1 + X + Y + \frac{{k_{cat}^{TE}p_{TE}X} + {{k_{cat}^{TV}\lbrack{TVMVP}\rbrack}Y}}{k_{dD}}}{{k_{cat}^{TE}p_{TE}} + {k_{cat}^{TV}\lbrack{TVMVP}\rbrack} + k_{dA}}},} & \left( 19^{*} \right)\end{matrix}$Where

$X \equiv {\frac{k_{cat}^{TV}\lbrack{TVMVP}\rbrack}{{k_{cat}^{TE}p_{TE}} + k_{d\; B}}\mspace{14mu}{and}\mspace{20mu} Y} \equiv \frac{k_{cat}^{TE}p_{TE}}{{k_{cat}^{TV}\lbrack{TVMVP}\rbrack} + k_{dC}}$

We used this equation to fit the experimentally observed bandpassbehavior (FIG. 3D).

Cell Line Construction

Some of the experiments do require more stable/homogenous transgeneexpression, for which the inventors used antibiotic selection togenerate cell lines with stably integrated transgenes. Two days aftertransfection in 24-well plates, cells were transferred to 6-well plateand selected with either 50 μg/mL Hygromycin (Hyg) or 400 μg/mLGeneticin (Gen). SOS_(CA) cells: CMV-TO-MSos-2A-H2BChe-FlpInco-transfected with pOG44, Hyg; pulse cells:PB-CMV-TO-rapTEV-teHCV-hcTVMV-tvDiTEV-Neo co-transfected with a plasmidexpressing PiggyBac transposase, Gen; EGFRvIII+ cells:PB-CMV-TO-EGFRvIII-IRES-nlsChe co-transfected with a plasmid expressingPiggyBac transposase, Gen. After PiggyBac-based integration, monoclonalcell populations were established through limiting dilution, andpreliminary screening was performed to identify clones with highesttransgene expression (based on GFP that serves as the scaffold in iTEV,and mCherry that's co-expressed with EGFRvIII), which were used insubsequent experiments. Among the pulse cell clones with highest GFPexpression, the one with the least variance was selected. The inventorsthen subjected this clone to another round of transgenesis (Hyg,CMV-TO-Cer-HO1-FlpIn co-transfected with pOG44) to provide Cerulean as asegmentation marker and heme oxygenase-1 to increase the intracellularconcentration of biliverdin that's necessary for enhancing iTEV signal.The final cell line was used in time-lapse imaging.

Time-Lapse Imaging

For time-lapse imaging of pulse dynamics (FIGS. 3A-3H) monoclonalpulse-generation cells were mixed with parental wild-type HEK293 cellsat a 1:10 ratio. Cells were plated on 24-well glass-bottom plates whichhad been coated with 5 μg/mL with hamster fibronectin for 1 hour at roomtemperature. Cells were induced with 100 ng/mL doxycycline overnight innormal culturing conditions. The following morning, the media wasreplaced with imaging media containing FluoroBrite DMEM (Thermo Fisher)supplemented with 10% FBS, 1 mM sodium pyruvate, 1 unit/ml penicillin, 1μg/ml streptomycin, 2 mM L-glutamine and 1×MEM non-essential amino acidsand 100 ng/mL doxycycline.

Time-lapse images were acquired on an inverted Olympus IX81 fluorescencemicroscope with Zero Drift Control (ZDC), an ASI 2000XY automated stage,iKon-M CCD camera (Andor, Belfast, NIR), and a 60× oil objective (1.42NA). Fluorophores were excited with an X-Cite XLED1 light source (LumenDynamics). Cells were kept in a custom-made environmental chamberenclosing the microscope, with humidified 5% CO2 flow at 37° C.Microscope and image acquisition were controlled by Metamorph software(Molecular Devices).

Imaging started approximately 2 hours after changing the media tofluorescent imaging media. 5 nM rapamycin was added after approximately2 hours of imaging to induce the pulse. Images were acquired every 20 or25 min, typically for 20-40 hrs. Cells that were in the field of viewbefore rapamycin induction and remained alive and visible in the fieldof view without death for at least 20 hours were used for initial dataanalysis.

For analysis, the inventors only included cells that remained alivethroughout the duration of the experiment, remained within the field ofview, and had detectable signal/background ratio. IFP fluorescenceintensity is dependent on the biliverdin chromophore. Addition ofexogenous biliverdin increases IFP fluorescence but also producesIFP-independent background fluorescence. For movies, to minimizebackground, the inventors omitted biliverdin from the media, relyinginstead on lower concentrations produced endogenously. Under theseconditions, IFP excitation illumination levels caused somephototoxicity, resulting in a subpopulation of ˜50% of cells that diedwithin ˜7 hours. The remaining cells continued active division until theend of the movie, or until exit from the field of view. These cellsexhibited a range of IFP fluorescence levels overlapping background.30-60% of these cells in which IFP fluorescence exceeded background.About half of this set had morphologies that were amenable toimage-based segmentation and therefore were analyzed further. Withinthis group, the inventors verified that the circuit dynamics wereindependent of expression level, as measured by peak IFP fluorescence,suggesting that circuit dynamics are not influenced by expression levelwithin this range, according to some embodiments.

Single-Cell Tracking and Image Normalization:

Single-cell tracking and image normalization procedures were performedas described herein. Briefly, cells constitutively express cytoplasmicCerulean as a segmentation marker. Due to the diffuse and weak Ceruleansignal, manual segmentation was frequently required and cell boundarieswere identified in part by phase contrast and GFP fluorescence images(GFP is the protein identified as the “split scaffold” in FIG. 3E. Itserves a structural role in the context of the IFP reporter, but alsofluoresces).

We performed image correction to account and correct for non-uniformillumination as well as background. The inventors assumed atime-independent spatially inhomogeneous illumination profile that ischaracteristic of the optical path, I(x,y). This was extracted byfitting the low intensity “non-cell” pixels in the images with a twodimensional paraboloid. In addition the inventors considered two sourcesof background fluorescence: First, the detector produces a basal pixelvalue even in the absence of light. This value, denoted B, is spatiallyhomogeneous and time-independent. Second, the inventors considered theautofluorescence of the media. This background source changes over time,and exhibits a spatial profile proportional to the illumination profile,A(t)*I(x,y). With these assumptions, the inventors extracted thecorrected fluorescence value using the following equation:

${F_{corrected}\left( {x,y,t} \right)} = {\frac{{F_{raw}\left( {x,y,t} \right)} - B}{I\left( {x,y} \right)} - {A(t)}}$

For generating a movie, mean intensities <5% were set to zero and meanintensities >99.5% were set to maximum pixel values to limit the effectof extreme pixel values due to noise on image brightness and contrastsettings.

Quantification of Amplitude and Pulse Decay:

Data Processing:

The amplitude and pulse decay calculations were based on total levels offluorescence in the IFP fluorescent channel. To systematically quantifythe fluorescent signal in the IFP channel, total IFP signal intensityIFP(x,y,t) was normalized by the total constitutive Cerulean signalCFP(x,y,t) and rescaled with a baseline variable (90th percentile of

$\frac{{IFP}\left( {x,y,t} \right)}{{CFP}\left( {x,y,t} \right)}$at all x positions.) To capture the pulse of IFP signal and avoiddistortion of the peak shape, the resulting data was smoothed with aSavitzky-Golay filter using a 3rd order polynomial and a window lengthof 9. After smoothing, the data were interpolated to equidistanttimepoints of 20 minute intervals (FIG. 3H).

Fitting:

Pulsing dynamics were fitted by taking the smoothed and interpolateddata and subtracting the minimum value of the normalized signalintensity from each timepoint. Using MATLAB's tfest function, thenormalized data were deconvolved with a finite impulse signal and athird-order linear transfer function resulting in the equation:y=a ₁ e ^(p) ¹ ^(x(t)) +a ₂ e ^(p) ² ^(x(t)) +a ₃ e ^(p) ³ ^(x(t))

The resulting fit was used to determine: (1) the location at which themaximal value of IFP occurred and (2) the delay time, τ, after peaksignal at which the signal intensity decayed to 50% its maximum value.After determining the peak location and τ, the mean and standarddeviation were calculated.

Example 2—Engineering of a Split HCV Protease

Hepatitis C Virus (HCV) protease had not, to the inventors' knowledge,been successfully split. The inventors used a strategy based on thefollowing criteria: (1) each fragment had to be predicted to be a foldedsubunit, (2) the location of the split between the two fragments had tooccur in a loop or unstructured region; and (3) the three residues ofthe catalytic triad could not be located on the same fragment. Theinventors engineered a split Hepatitis C Virus (HCV) protease mutantssatisfying these criteria and tested their catalytic activity by rescueof a degron-tagged fluorescent protein relative to a non-rescuedfluorescent protein control. Based on initial results, the inventorsfurther tested additional split sites by shifting the position at whichwe split the two fragments by one residue in both the N and C terminaldirection. The following split sites exhibited the best performance:sHCV 120 and sHCV 122. See Wehr et al, Monitoring regulatedprotein-protein interactions using split TEV. Nat. Methods 3, 985(2006), for some materials and methods relating to this methodology.

Example 3—Alternative Designs for Protease Activated Proteases

Described below are some additional embodiments of compound proteases,and resulting data when the designs were tested.

Design: HCV protease is tagged with an auto-inhibitory domain that canbe removed with TEV protease (FIG. 12A). Result: This domain included amutated HCV cleavage site sequence that binds to, but is no longercleaved by, HCV protease. Adding TEV protease (lower panel) did notincrease HCV protease activity compared to a control lacking TEVprotease (upper panel). Plots represent flow cytometry distributions ofthe Cit-hcvs-DHFR reporter.

Design: HCV protease is tagged with a DHFR degron that can be removedwith TEV protease (FIG. 12B). Result: Adding TEV protease did notincrease HCV protease activity, as indicated by a Cit-hcvs-DHFRreporter.

Design: Split TEV protease is tagged with a degron (four tandem repeatsof ubiquitin, because the typical DHFR degron is even less effective) onthe end of one of the leucine zippers, and the degron is removable byTVMV protease (FIG. 12C). Result: The degron did not fully repress TEVprotease activity, and adding TVMV protease only led to a small increaseof TEV protease activity, as indicated by a Cit-tevs-DHFR reporter.However, this design was at least partially successful because somepositive regulatory effect was seen.

Design: The N-terminal half of TEV protease is caged with acomplementary leucine zipper and a catalytically inactive C-terminalhalf, and the caging domains are removable with TVMV protease (FIG.12D). Result: The caging domains did not fully repress TEV proteaseactivity, and adding TVMV protease did not increase TEV proteaseactivity, as indicated by a Cit-tevs-DHFR reporter.

Example 4—Applications

Some applications of some embodiments of the systems, methods and/orcompositions provided herein include the following:

-   -   Kill switches: the ability to kill engineered cells in response        to a signal is useful for many emerging cell based therapies.        Existing methods may produce toxicity or cell death when not        desired, i.e. in the absence of the kill switch input. Some        embodiments include the construction of kill switches in which        such effects can be suppressed through a protease-based        reciprocal inhibition motif or through feed-forward loop        structures.    -   Virally delivered synthetic circuits: some embodiments include        the ability to deliver complex programmed protein-level        functions into cells using a variety of non-integrating vectors.        This capability may avoid potential mutagenesis that occurs with        gene regulation based systems.    -   Oncolytic viral therapies: by encoding protein level circuits on        oncolytic viruses, one may deliver functions that specifically        kill or inactivate tumor cells conditionally depending on the        tumor cells' state(s).    -   Gene drive payloads: gene drive technology may enable efficient        super-Mendelian propagation of genetic systems within mating        populations of organisms. Gene drive applications of the current        embodiments are enabled by the disclosure herein based on the        ability to package sophisticated functions in compact genetic        systems. Some embodiments of the systems described herein can        enable gene drive payloads that perform such functions. For        example, for insect vector control, a protein-circuit that        specifically kills mosquitoes infected by human pathogens such        as Dengue virus.    -   Cell type specific control of cell fate. Regenerative medicine        sometimes requires precise manipulation of cell fate. Protein        level circuits can be transiently introduced into cells to        control the activation of fate regulating genes and thereby        induce specific cell fates. This activity can be coupled to        modules within the circuit that detect the state of the cell and        make cell fate control conditional on cell state. This may avoid        a problem of activating the same genes in a heterogeneous cell        population, and also may avoid permanent genetic modification.    -   Extracellular protein level feedback circuits that control blood        clotting. The system described herein enables protein circuits        that function outside cells to detect blood clots or        pathological conditions and trigger clot removing functions.    -   Subcellular functions. The disclosure herein enables protein        circuits that function in specific subcellular compartments or        sites. Circuits can operate to modulate the behavior of specific        synapses or organelles such as mitochondria.    -   T-Cell therapies. The disclosure herein enables protein circuits        that function when in the presence or absence of ligand on an        adjacent cell. Circuits can operate to control activity of        T-cells, which may express a chimeric antigen receptor. The        circuit can enable tuning and/or control of the receptor        activity, and can also function to integrate multiple internal        and external signals into a response.

These are only some examples of the many applications of the systems,methods and compositions provided herein.

The term “comprising” as used herein is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps.

The above description discloses several methods and materials of thepresent invention. This invention is susceptible to modifications in themethods and materials, as well as alterations in the fabrication methodsand equipment. Such modifications will become apparent to those skilledin the art from a consideration of this disclosure or practice of theinvention disclosed herein. Consequently, it is not intended that thisinvention be limited to the specific embodiments disclosed herein, butthat it cover all modifications and alternatives coming within the truescope and spirit of the invention.

All references cited herein, including but not limited to published andunpublished applications, patents, and literature references, areincorporated herein by reference in their entirety and are hereby made apart of this specification. To the extent publications and patents orpatent applications incorporated by reference contradict the disclosurecontained in the specification, the specification is intended tosupersede and/or take precedence over any such contradictory material.

All titles, headings and subheadings used herein are meant to addadditional disclosure of some embodiments, but are in no way limitingwith regard to the subject matter contained anywhere herein.

What is claimed is:
 1. A system comprising: a first protease; a secondprotease; and one or more target proteins each comprising: a firstdegron of the target protein that destabilizes the target protein whenpresent on the target protein by enhancing degradation of the targetprotein, a cut site specific for the first protease between the degronof the target protein and a part of the target protein, wherein thetarget protein is configured to be stabilized by cleavage of its cutsite specific for the first protease, another degron of the targetprotein, and a cut site specific for the second protease connected tothe other degron of the target protein, wherein the target protein isconfigured to be destabilized by cleavage of the cut site specific forthe second protease regardless of whether the first degron of the targetprotein is present on the target protein.
 2. The system of claim 1,wherein the other degron of each target protein comprises an N-enddegron that is conditional on cleavage of the cut site specific for thesecond protease.
 3. The system of claim 1, further comprising a thirdprotease comprising a cut site specific for the second protease, whereinthe third protease is configured to be deactivated by cleavage of itscut site specific for the second protease; and wherein the secondprotease comprises a cut site specific for the third protease, whereinthe second protease is configured to be deactivated by cleavage of itscut site specific for the third protease.
 4. The system of claim 1,wherein the second protease further comprises a first domain of thesecond protease, a second domain of the second protease, a firstcomplementary association domain, and an optional second complementaryassociation domain of the second protease connected to the first orsecond domain of the second protease; wherein the first domain of thesecond protease comprises the cut site specific for the third protease;wherein the second domain of the second protease comprises another cutsite specific for the third protease; wherein the first complementaryassociation domain of the second protease optionally comprises two partsof the complementary association domain of the second protease, eachpart of the complementary association domain of the second proteaseconnecting to one of the second protease's cut sites specific for thethird protease; and wherein the second protease is configured to bedeactivated by cleavage of either of its cut sites.
 5. The system ofclaim 1, wherein the third protease further comprises an optionalassociation domain of the third protease, and wherein cleavage of thethird protease's cut site by the second protease removes at least partof a cleavage domain of the third protease, thereby deactivating thethird protease.
 6. The system of claim 1, wherein the stability of thetarget proteins comprises an analog behavior that is dependent on aconcentration of the first protease, wherein a higher concentration ofthe first protease has a greater stabilizing effect on the targetproteins than a lower concentration of the first protease.
 7. The systemof claim 1, wherein the stability of the target proteins comprises ananalog behavior that is dependent on a concentration of the secondprotease, wherein a higher concentration of the second protease has agreater destabilizing effect on the target proteins than a lowerconcentration of the second protease.
 8. The system of claim 7, whereinthe concentration of the second protease is decreased by a higherconcentration of the third protease as compared to a lower concentrationof the third protease or by a higher amount of a nucleic acid encodingthe third protease as compared to a lower amount of a nucleic acidencoding the third protease.
 9. The system of claim 7, wherein theanalog behavior of the target protein that is dependent on aconcentration of the second protease is more sharp and/or comprises agreater threshold for destabilizing the target protein at a higherconcentration of the third protease as compared to a lower concentrationof the third protease, or at a higher amount of a nucleic acid encodingthe third protease as compared to a lower amount of a nucleic acidencoding the third protease.
 10. The system of claim 1, wherein thefirst protease further comprises a first domain of the first proteaseand a second domain of the first protease; wherein the first domain ofthe first protease connects to a first conditional dimerization domainof the first protease; wherein the second domain of the first proteaseconnects to a second conditional dimerization domain of the firstprotease; wherein the first and second conditional dimerization domainsof the first protease are configured to dimerize with each other uponbinding a dimerizing agent.
 11. The system of claim 10, wherein theconditional dimerization domains of the first protease each comprise oneof an FK506 binding protein (FKBP), GyrB, GAI, Snap-tag, eDHFR, BCL-xL,CalcineurinA (CNA), CyP-Fas, FRB domain of mTOR, GID1, HaloTag, and/orFab (AZ1).
 12. The system of claim 10, wherein the dimerizing agentcomprises FK1012, FK506, FKCsA, Rapamycin, Coumermycin, Gibberellin,HaXS, TMP-HTag, or ABT-737.
 13. The system of claim 1, wherein the firstprotease comprises tobacco etch virus protease (TEVP).
 14. The system ofclaim 1, wherein the second protease comprises tobacco vein mottlingvirus protease (TVMVP).
 15. The system of claim 1, wherein one targetprotein of the one or more target proteins comprises Citrine.
 16. Thesystem of claim 1, wherein one target protein of the one or more targetproteins comprises, from the N-terminal to the C-terminal of the onetarget protein, an N-end degron as the other degron comprising a tobaccovein mottling virus protease (TVMVP) cut site specific for the secondprotease, Citrine, a tobacco etch virus protease (TEVP) cut site as thecut site specific for the first protease, and a non-N-end degron as thefirst degron.